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"Hello Miss, I MEAN sir, I MEAN Ma'am, I MEAN sir"
"Why hello!"
Androgyny is the concept that someone can be between genders and/or inlabelable and/or has aspects that can be attributed to the opposite sex. Brilliant really, you can go into any toilet you want and not bat an eyelid. An Androgynous appearance can indicate one of two things. One, that a person considered him/her/hir self to be between genders, that no label can be attached to him/her/hir, or that they just really REALLY like to confuse people.
How to spot an androgyn[edit]
Look at the person next to you. Can you tell what sex s/he is for sure? And I don't mean a passable transsexual, I mean REALLY you don't have a fookin' clue weather this person is male or female. NO! DON'T ASK IF S/HE'S A GUY OR A GIRL! S/he'll beat you to within an inch of your life.
Right, just watch him/her/hir. Look at the way s/he behaves, ok and...OH, s/he has a girl/boyfriend, but could s/he be a lesbian? I have no idea!!!! Right, s/he's going to the toilet ok...ok...ummm, DAMN IT IT'S A UNISEX TOILET! How can you tell what s/he is?!!! Right, s/he's come out the toilet. Now wha...F---/he knows I'm looking. S/he's given me the finger, damn s/he's gone.
What to do when you meet an androgyn[edit]
First thing you must not do is ask 'Are you a dude or a girl', as s/he will either confuse you or beat you vigorously. You may talk to the androgyn but whatever you do DON'T MENTION THE ANDROGYNY.
Also, don't believe that you won't meet an androgyn, because YOU WILL, really, you will! Trust me!
Androgynous identities[edit]
For those who identify as androgynous, there are many of those who identify with terms within this large umbrella term. Before I start, imagine a spectrum with the most eye crushingly masculine man possible at one end, and the most Effeminate woman at the other end. Please take into mind though that these are just the perceived understandings of one rambling idiot and may not be how things are for most people (Well this is uncyclopedia after all). Ready?
- Gender Neutral (neutrois): Where one's gender identity is standing in the center, one foot on the male half, the other on the female half.
- Bi-gender: Where one's gender identity would be neutral, but would swing from time to time from male to female juuuuuust slightly.
- Gender fluid: Where one's gender identity would run up and down the spectrum like a hyperactive child on Red Bull.
- Genderqueer: Where the person's gender identity flips the bird at the gender spectrum and goes off to play with the light spectrum.
- Genderless: Where a person's gender identity doesn't appear on the gender spectrum and just sits at home reading manga.
- Polygender: Where a person's gender clone's itself and goes to two separate points on the spectrum.
Why Androgyny[edit]
There are several reasons for androgyny. These range from an expression of gender identity that is primarily neutral, or the opposite to his/her sex or because s/he F---ing CAN! Nine out of ten times it's the former.
Famous androgyns[edit]
- Oscar Wilde
- The Stig (Lets face it, we don't know what he is).
- You
- Michael Jackson (Note, Not all Androgyns are eccentric pedofiles, just Michael).
- Brian Molko
- Tedd of El goonish shive fame...
- Scott of Ctrl alt del fame...
- infact...
- Think of any webcomic.
- There's bound to be a androgyn there somewhere....
- Any anime, manga, or Japanese video game is bound to have an androgyn as well. This, however, is known as "bishounen," which is Japanese for "Androgynous Boy," so these characters are almost always male.


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WHAT: A research team at the National Institutes of Health has found that bacteria that normally live in the skin may help protect the body from infection. As the largest organ of the body, the skin represents a major site of interaction with microbes in the environment. Although immune cells in the skin protect against harmful organisms, until now, it has not been known if the millions of naturally occurring commensal bacteria in the skin—collectively known as the skin microbiota—also have a beneficial role. Using mouse models, the NIH team observed that commensals contribute to protective immunity by interacting with the immune cells in the skin. Their findings appear online on July 26 in Science.
The investigators colonized germ-free mice (mice bred with no naturally occurring microbes in the gut or skin) with the human skin commensal Staphylococcus epidermidis. The team observed that colonizing the mice with this one species of good bacteria enabled an immune cell in the mouse skin to produce a cell-signaling molecule needed to protect against harmful microbes. The researchers subsequently infected both colonized and non-colonized germ-free mice with a parasite. Mice that were not colonized with the bacteria did not mount an effective immune response to the parasite; mice that were colonized did.
In separate experiments, the team sought to determine if the presence or absence of commensals in the gut played a role in skin immunity. They observed that adding or eliminating beneficial bacteria in the gut did not affect the immune response at the skin. These findings indicate that microbiota found in different tissues—skin, gut, lung—have unique roles at each site and that maintaining good health requires the presence of several different sets of commensal communities.
This study provides new insights into the protective role of skin commensals and demonstrates that skin health relies on the interaction of commensals and immune cells. Further research is needed, say the authors, to determine whether skin disorders such as eczema and psoriasis may be caused or exacerbated by an imbalance of skin commensals and potentially harmful microbes that influence the skin and its immune cells.
The study was led by investigators in the laboratories of Yasmine Belkaid, Ph.D., at the National Institute of Allergy and Infectious Diseases, in collaboration with Julie Segre, Ph.D., at the National Human Genome Research Institute, and Giorgio Trinchieri, M.D., and Heidi Kong, M.D., at the National Cancer Institute. All three Institutes are NIH components.
S Naik et al. Compartmentalized control of skin immunity by resident commensals. Science DOI: 10.1126/science.1225152 (2012).
Yasmine Belkaid, Ph.D., chief, Mucosal Immunology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, and Julie Segre, Ph.D., senior investigator, Genetics and Molecular Biology Branch, National Human Genome Research Institute, are available for comment.
To schedule interviews, please contact Julie Wu, (301) 402-1663, email@example.com, or Raymond MacDougall, (301) 402-0911, email@example.com.
NIAID conducts and supports research—at NIH, throughout the United States, and worldwide—to study the causes of infectious and immune-mediated diseases, and to develop better means of preventing, diagnosing and treating these illnesses. News releases, fact sheets and other NIAID-related materials are available on the NIAID Web site at http://www.niaid.nih.gov.
NHGRI is one of the 27 institutes and centers at the National Institutes of Health, an agency of the Department of Health and Human Services. The NHGRI Division of Intramural Research develops and implements technology to understand, diagnose and treat genomic and genetic diseases. Additional information about NHGRI can be found at its website, www.genome.gov.
NCI leads the National Cancer Program and the NIH effort to dramatically reduce the burden of cancer and improve the lives of cancer patients and their families, through research into prevention and cancer biology, the development of new interventions, and the training and mentoring of new researchers. For more information about cancer, please visit the NCI Web site at http://www.cancer.gov or call NCI's Cancer Information Service at 1-800-4-CANCER (1-800-422-6237).
About the National Institutes of Health (NIH): NIH, the nation's medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit http://www.nih.gov/.
NIH…Turning Discovery Into Health

subset: commoncrawl


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DETROIT >> Tyler Collins got a chance to win Monday's game, as a pinch runner.
Now he's getting a chance to make his first big-league start, against a left-hander.
Tough spot to put the rookie, who is also left-handed?
Not necessarily.
"I do think he can handle it. He's tough enough mentally to handle facing a left-handed pitcher. I don't know ideally if that would be the first spot I would put a guy, but I don't dislike him against (Royals starter Jason) Vargas," manager Brad Ausmus said. "He knew on Monday after the game. He's probably sleepless. He probably hasn't slept in two days."
While the other primary left fielder, Rajai Davis, is fine, there's are more reasons Ausmus thought it might be a good matchup.
One, Davis is 1-for-11 career against Vargas.
Two, Vargas has allowed a higher batting average against left-handed hitters over the last few years; lefties hit .327 off Vargas last year.
Three, Collins hit .289 with a .953 OPS vs. left-handed pitchers last year at Double-A Erie, with 11 home runs. He hit .219 vs. right-handers with 10 homers, in roughly 40 more at-bats.
It should also help Collins to be hitting in the No. 2 hole, ahead of Miguel Cabrera, where he might get a few more strikes to hit.
"That's the theory. That's what we're hoping. I'm sure Tyler will be excited," Ausmus said. "The hardest part for a young player is to control the emotions and kind of slow the game down in his mind. But hopefully hitting in front of Miggy gives him the opportunity to get a few more pitches."
Putting Collins in the No. 2 spot in the order displaces Torii Hunter, who hit there all of last season, as well as in the opener.
"I think Torii, especially against left-handed hitters, is one guy that slots in well in the fifth spot," Ausmus said. "The five hole for us, we don't really have a prototypical five-hole hitter. But I think against a left-handed pitcher, Torii looks more like a five-hole hitter than anyone else on the team."


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Though I may continue to update this blog on a less frequent basis, I wanted to alert all of you to check out my new blog over at www.zachhellermarketing.com.
I've found a new home on the Squarespace platform which has allowed me to reinvigorate my passion for blogging and for marketing. I'll be updating the blog frequently with information about marketing, small business, etc., similar to the material that has brought you here.
I encourage you to check it out, "I" of the Consumer, and subscribe today!


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Trinity hockey doubled up by West Haven
STAMFORD -- Bad bounces, deflections and other lucky plays happen all the time in ice hockey. Rarely, however, does one such play lead to three goals.
It seems that's what happened in West Haven High's 4-2 boys hockey victory over Trinity Catholic Wednesday night at Terry Conners Rink.
"We caught a lucky break at the end of the second period and it helped us in the third period, too," West Haven coach Joe Morrell said.
West Haven's Joe Ayala was credited with the goal off a soft shot that was deflected into the Trinity net by a Crusader defenseman. The goal came with just 30 seconds remaining in the second period and gave the visiting Blue Devils a 2-1 lead. Less than five minutes into the third period the West Haven lead had ballooned to 4-1.
"We fought back to tie it (at 1) and then they score on a deflection," Trinity coach Ken Smith said. "It certainly took the wind out of our sails."
West Haven outshot the Crusaders 7-1 in the first five minutes of the third period and Peter Lynn's unassisted goal 2:47 into the stanza made it 3-1. Two minutes later Jim Morrissey scored off a great pass from Austin Hanson to make it 4-1. The three-goal flurry helped give the Blue Devils (1-2) their first win while leaving the Crusaders (0-3) winless.
"I'm not ecstatic with our 0-3 start but I'm happy with our kids' effort tonight," Smith said. "That's a good team and I saw a lot of positive things. We'll probably give a lot of teams problems down the road."
If the Crusaders do that, it will be similar to how last season played out. The Crusaders, who lost 4-2 to Glastonbury and 3-2 to Fermi-Enfield to open this season, started 2-10 in 2011-12 before winning eight of nine to finish 10-12.
A few of the positive signs were from Eryk Wielgos, who scored the Crusaders' first goal off a faceoff to tie it at 1 with 5:39 remaining in the second period. And Will Egan's power play goal with 6:19 left to play -- off a beautiful pass from Steve Lambrinakos -- was a work of art.
Meanwhile, Trinity goalie Kyle Odierno was strong in net, stopping 30 of the 34 shots he faced. He had no chance on the deflected goal, or on Morrissey's tally that made it 4-1.
West Haven, which lost to Greenwich 4-2 and Darien 5-1 before Wednesday, is coming off a 15-8 season. The Blue Devils, however, graduated nine players and have only two returning seniors.
"I knew it would be a good game because at 0-2, both teams needed it," Morrell said. "We've been struggling scoring so maybe that lucky goal will get us started. It sure did tonight."
Hanson opened the scoring for West Haven in the opening minute of the second period. Jaeson Puleo added two assists and goalie Mike Savino stopped 23 of 25 shots.
West Haven 4, Trinity 2
Records: West Haven 1-2, Trinity 0-3.
Goals: WH--Austin Hanson, Joe Ayala, Peter Lynn, Jim Morrissey; T--Eryk Wielgos, Will Egan. Assists: WH--Jaeson Puleo 2, Morrissey, Hanson; T--Carmine DeRubeis, Sean Dolan, Steve Lambrinakos. Goalie saves: WH--Mike Savino 23; T--Kyle Odierno 30.


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EXCLUSIVE! Will There Be A 'Full House' Reunion On 'Glee?'
Now that John Stamos (AKA Uncle Jessie) is on Glee, is it time to see Stephanie Tanner and the rest of the gang from Full House finally give fans what they want, a full-fledged reunion — and Jodie Sweetin says she's completely on board!
Everywhere you look, there's a heart, a hand to hold on to. Everywhere you look, there's a face of somebody who needs you — and the faces we need most are Danny, Joey, Jesse, D.J, Stephanie and Michelle! HollywoodLife.com caught up with Jodie Sweetin (the one and only, Stephanie Tanner) who told us she's vying for a Full House reunion on the hit show Glee now that her former co-star John Stamos is a recurring character! How awesome would that be?
"I would love to do something like that! I mean Glee looks like a ton of fun, I actually went to High School with Matt Morrison, we did musical theater together," Jodie, 28, told us exclusively at the Radd Rewards in Downtown LA Oct.14 adding, "But you know, people have wanted a Full House reunion for years, there is always the talk of it, and if it was done on Glee that would be lots of fun."
Just imagine the whole cast of Full House and Glee busting out in the Full House theme — or better yet, Mary Kate & Ashley Olsen hooking up with Cory Monteith's character, Finn! Seriously, the potential for epic-ness would be unlimited.


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Eastern Wake News
Wake County leaders agree on school construction deal
Wake County commissioners will give up their efforts to take over control of school construction as part of an agreement Thursday to work more closely with the school board on how schools are located and built.
Under the deal reached Thursday by the leaders of both boards, the school board will retain authority over school construction but will involve the Board of Commissioners more in the planning process. In addition, the leaders agreed that they'll ask state legislators not to act on a pending bill that would have turned over school construction authority in Wake to the Board of Commissioners.
"This is a good day for Wake County," school board member Keith Sutton said.
The agreement appears to resolve a more than decadelong dispute between Wake County's two ruling boards over who will have the last say on planning and building schools. It comes as the school system works through finding building sites and planning the construction and renovations to be funded by the $810 million school-construction bond issue approved by voters in October.
The school board is scheduled to vote on the agreement Tuesday. The Board of Commissioners is scheduled to take it up March 17.
The commissioners turned to the General Assembly for help last year to take control of school construction. School leaders have insisted that the district had the expertise needed to build schools.
A bill giving Wake County commissioners control over school construction passed the state Senate but was rejected by the House in July. It's eligible for reconsideration when the General Assembly reconvenes in May.
Both the Republican-led commissioners and Democratic-led school board had hired lobbyists last year to oppose each other on the bill. But leaders of both boards said the letters they'll write to legislators after the agreement is approved should cause them to drop any action on the bill.
'Good faith effort'
The agreement has wording saying it "reflects a good faith effort to resolve past issues and conflicts amicably and locally." It also says neither side will be "taking any measures to alter the respective legal rights and duties of either party."
"We've resolved it locally," said school board Chairwoman Christine Kushner. "It's an important show of good faith. I don't know how anyone can ignore that."
Tony Gurley, vice chairman of the Board of Commissioners, said it wouldn't make sense for the General Assembly to pass the school construction bill this year because members won't have had the time to see how the agreement has turned out. He said that after a few years, commissioners will be able to see whether they should return to the legislature to ask for the construction authority.
The agreement allows either side to back out after giving 30 days' notice.
Under the agreement, the commissioners and school board will form a "joint school facilities core team" consisting of school and county staff members. The team will develop a process for how both boards can work together on the research, investigation and evaluation of acquiring school sites, and designing and construction of schools.
The agreement also says the team will regularly keep both boards informed on construction issues. One of the complaints from commissioners is that they don't get information soon enough on construction projects, resulting in them voting to delay or reject items.
"It will give you, the school board, some assurance that you won't be surprised if we turn down something," Gurley said.
The agreement also includes wording saying the school board can choose to turn over authority for individual construction projects to the Board of Commissioners.
Both panels portrayed the agreement as a victory for their sides.
"Yes, the school board retains the authority to build and renovate schools," Gurley said. "But we will get information sooner. It's a good compromise."
Gurley, who first proposed the agreement in November, said he didn't think it would have been approved if not for the threat of the legislation.
But Kushner stressed that the agreement maintains the current system in which school boards have control over school construction, but have to request funding from the Board of Commissioners.
"I think the legislation was just one-sided," Kushner said. "We believe in checks and balances, and I believe this agreement respects that."
Gurley said the agreement "benefits the taxpayers of Wake County." But Ed Jones, chairman of the Wake County Taxpayers Association, wasn't so sure. Jones said he was disappointed that the commissioners are giving up on seeking control over school construction.
"I still feel strongly that the county and the county commissioners have more ability to work with the building of schools," Jones said.


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Coffee Website Template
Template ID #62130 was created by our webmaster designer Jetimpex
The coffee theme found on this page utilizes the Bootstrap framework to make it easy develop a responsive theme that you can use on a regular website, tablet, or smart phone. Bootstrap was created by twitter to quickly boost a website's design and functionality. It provides a standard set of CSS and HTML conventions including typography, forms, buttons, tables, grids, and more. The Bootstrap framework consists of both JavaScript and CSS to provide these features, and included with that are special plugins such as default page types including under constructions, intro page, and 404 pages, portfolio tools, image sliders, social sharing plugins, and numerous CSS3 tricks such as animated hovers and buttons. This coffee website template is also known as template 62130 and will fill a width of up to 1920 pixels. It has had a total of 20 downloads since it was created by Jetimpex.
Main Page
Sources Included and Software Required
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Other places to find similiar Themes
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Jetimpex's Coffee Website Template 62130 resides in the Cafe & Restaurant and Most Popular categories so you will likely find additional layouts there that would fascinate you.


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Journal Article
© Jul 2005 Volume 3 Issue 1, Editor: Charles Despres, pp1 - 64
Look inside Download PDF (free)
Contrary to the "one‑fits‑all" approach used in the literature on how to sustain virtual communities of practice (VCoPs), this paper advocates that successful management practices should be contingent upon their basic characteristics. More specifically, this study of eight virtual communities of practice investigates how the actions taken by the communities' leadership teams may influence their success. The results show that decisions regarding the operational leadership of a VCoP are crucial elements to counteract the challenges arising from its structuring characteristics. Among those decisions, the choice and availability of a leader and the support of a coach are shown to be crucial.
Keywords: Virtual community of practice, virtual group, leadership, knowledge sharing, organizational learning
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It's called the Big Chill Bar and although it's located in the laid-back Florida Keys, a couple of their customers apparently need to learn a thing or two about the art of chillin' out.
Deputies were called to Jimmy Johnson's Big Chill Bar in Key Largo late Thursday night after a fight broke out, according to a news release from the Monroe County Sheriff's Office.
A Key Largo couple, along with their 21-year-old daughter and her pal, had been eating a pizza at the bar when a couple from Miami walked up and tried to swipe some of the pizza from the box, the release stated.
This led to a clash -- especially after the Miami couple, George Joseph Burns, 44, and Midiala Siverio Dunn, 43 – began calling the two girls "fat," according to the Sheriff's Office.
Things deteriorated rapidly when Burns then allegedly struck both girls, knocking one to the ground, deputies reported.
After Burns and Dunn had reportedly left the bar, they were confronted in the parking lot by one of the girl's dad who decided to weigh in on the ugly scene. Both men then began fighting, according to the Sheiff's Office.
When a concerned woman in the parking lot approached Dunn, who was standing near the two fighting men, to tell her to move out of harm's way, Dunn allegedly punched her.
Several witnesses corroborated the story told by the Key Largo couple, deputies stated.
After Burns and Dunn were both treated for injuries at a hospital they were transported to the The Big Cooler for some serious chilling-out time.
Burns is charged with three counts of battery; Dunn is charged with one count of battery.
And that's why, readers, pizza delivery is so popular.


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Alliance Theatre Company
Location: alliance theatre company
July 16, 2013
Taneisha Scott '16 of Baltimore spent her summer sharing musical theatre with children Atlanta.
Scott worked at the Alliance Theatre Company in the musical theatre education department for the summer in 2013. The internship included working with 15-22 children in musical theatre for various one-week camps. Scott enjoyed the experience and felt it really helped her to determine that she does want to work with children. She recently declared a music major and drama minor, and this internship reaffirmed that as the correct decision.
Last modified on Jul. 18th, 2013 at 10:10am by .


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19 July, 2012
In other words, we know quite well what's going on around the world. Some of those things are rejected by our national consciousness and some are considered worth emulating. Room-mating & Shacking-up, for instance, which is a regularlife-style among the American and European youth, has not yet become popular here. There should be plenty of reasons, justifying our refusal (I think only temporary) from this model of behavior. In the West it is natural for the opposite sexes (and not only) to shack up. Room-mating is totally ubiquitous in that part of the world – not here! Roommates in the West are financially independent in most cases. There are some parents of course who are not really loath to help out the gone-from-home kids, but this is not terribly popular. Financial independence among young men and women in Georgia is not as commonplace as one would desire. And even if it were, shacking up would not be welcome by most of the Georgian parents, especially the girls' moms and dads. Regardless certain sexual liberation, local tradition of nursing the family would not readily allow a young male and female to room-mate or shack-up. I am not saying that the country is totally devoid of such examples, but the number of room-mating couples is way below the Western level. Please don't get me wrong – I am not saying whether room-mating and shacking up are a good thing or a bad one. That's not for me to judge. This should rather be a sociologist's bread-and-butter. I am just making a simple statement: Georgia is still in the process of maturing for accepting room-mating and shacking-up as the way of life for young people. If I were asked a question about my daughter's shacking-up with her boy-friend, I would not be equipped with a prompt answer. I would probably have not-so-helpful second thoughts about the issue. Yes, it would be an issue for me. And I will have an attitude. I can't say why but I know I will. Something deep down in me is saying that we as a nation are not yet ready for this kind of life-style. You see, I can chip in several serious and not too serious reasons to validate my reservations: money, image, uneasy feelings, tradition, gossip, askance looks cast on my daughter and me, awkwardness of situations, various formalities, the young man's visitations, etc. This might sound funny, but that's the way most of us are in this country. That's why this belated perpetuation of room-mating and shacking-up as models of premarital cohabitation is becoming a norm here. Premarital sex is still causing certain social and personal pains in Georgia. But the flip side of the medal is that room-mating and shacking-up could have helped the increase of population in Georgia. I have said it before and I would like to reiterate: human reproduction should probably be a strategic direction of thought and behavior for this nation. Could this be enough reason to propagate room-mating and shacking-up among our young men and women? Who knows, but I am more likely to believe so than not!


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Do you really want to send decoy hunches a message saying you like their work?
Avril 14th is a track by Aphex Twin off the 2001 double CD (or double-double vinyl) release Drukqs. Like much of Aphex Twin's oeuvre, this song seems made to be used as part of a film score.
If you like Aphex Twin's ambient works, then you will love
this song. Note that this song is only indicative of maybe half of
Drukgs—the entire album alternates between drum and bass and softer
stuff like this, done with a prepared piano, piano, player piano or
keyboards. And loops.
If you have seen the films Marie Antoinette, Four Lions, Sleep Furiously or the SNL short Iran So Far—or heard that one song by Kanye West—than you mayhaps took notice of this calm & calm-ing bit of piano and wondered what it was. Or maybe not, as this is the sort of music that may be heard without registering if used as part of a film score.
Avril is French for April. The subtitle for this track is "The Kernow Festival" and refers to a celtic tradition celebrating Easter for families in Brittany and Cornwall. In the year in which Drukqs was released, Easter was on April 15th. So this song was either made on or for Holy Saturday. And loops.
Incidentally, April 14th is the birthday of BookReader, Daniel Clowes, Philip III, Adrien Brody, Bruce Sterling, Ritchie Blackmore, Ali Akbar Khan and my sister. And loops.
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Principles of RACE
Rapid Amplification of cDNA Ends (RACE) is a procedure for amplification of nucleic acid sequences from a messenger RNA template between a defined internal site and either the 3´ or the 5´ end of the mRNA (1). This methodology of amplification with single-sided specificity has been described as "one-sided" PCR (2) or "anchored" PCR (3). PCR requires two sequence-specific primers that flank the sequence to be amplified (4,5). However, to amplify and characterize regions of unknown sequences, this requirement imposes a limitation (3). 3´ RACE takes advantage of the natural poly(A) tail found in mRNA as a generic priming site for PCR. In this procedure, mRNAs are converted into cDNA using reverse transcriptase (RT) and an oligo-dT adapter primer. Specific cDNA is then amplified by PCR using a gene-specific primer (GSP) that anneals to a region of known exon sequences and an adapter primer that targets the poly(A) tail region. This permits the capture of unknown 3´-mRNA sequences that lie between the exon and the poly(A) tail. 5´ RACE uses an antisense gene specific primer for the synthesis of specific cDNA by reverse transcriptase.
Prior to PCR, a TdT-tailing step attaches an adapter sequence to the unknown 5´ sequences of the cDNA. Specific cDNA is then amplified by PCR using a GSP that anneals in a region of known exon sequences and an adapter primer that targets the 5´ terminus. RACE has been used for amplification and cloning of rare mRNAs (6) and may be applied to existing cDNA libraries (7). Additionally, RACE products can be directly sequenced without any intermediate cloning steps (8,9), or the products may be used to prepare probes (10). Products generated by the 3´ and 5´ RACE procedures may be combined to generate full-length cDNAs (10,11). Lastly, the RACE procedures may be utilized in conjunction with exon-trapping methods (12) to enable amplification and subsequent characterization of unknown coding sequences.
Summary of the 3´ RACE System
The 3´ RACE procedure is summarized as follows. First strand cDNA synthesis is initiated at the poly(A) tail of mRNA using the adapter primer (AP). After first strand cDNA synthesis, the original mRNA template is destroyed with RNase H, which is specific for RNA:DNA heteroduplex molecules. Amplification is performed, without intermediate organic extractions or ethanol precipitations, using two primers: one is a user-designed GSP that anneals to a site located within the cDNA molecule; the other is a universal amplification primer that targets the mRNA of the cDNA complementary to the 3´ end of the mRNA. Two universal amplification primers are provided with the system. The universal amplification primer (UAP) is designed for the rapid and efficient cloning of RACE products using the uracil DNA glycosylase (UDG) cloning method (13–16). The abridged universal amplification primer (AUAP) is homologous to the adapter sequence used to prime first strand cDNA synthesis.
Since the 3´ RACE System utilizes the poly(A) tail region as an initial priming site, multiple amplification products may be synthesized, depending on the degree of specificity conferred by the GSP. To generate a specific amplification product, the user may find it advantageous to design a second "nested" GSP, as recommended by Frohman et al. (10) and reamplify the RACE products; this procedure is discussed in greater detail at the end of this chapter
Isolation of Total RNA
One of the most important factors preceding the synthesis of substantially full-length cDNA is the isolation of intact RNA. The quality of the RNA dictates the maximum amount of sequence information that can be converted into cDNA. Thus, it is important to optimize the isolation of RNA from a given biological source and to prevent adventitious introduction of RNases (17) and inhibitors of reverse transcriptase such as guanidinium salts, SDS and EDTA (18). RNA can be isolated using a variety of methods. The recommended method for 3´ RACE is the guanidine isothiocyanate/acid-phenol method originally described by Chomzynski and Sacchi (19). The TRIzol Reagent method is an improvement of the original single-step method of Chomczynski and Sacchi (20) and can be used for the preparation of RNA from as little as 103 cells or milligram quantities of tissue (21). Total RNA isolated with Invitrogen TRIzol Reagent is undegraded and essentially free of protein and DNA contamination. For the isolation of RNA from small quantities of sample (<106 cells or <10 mg tissue) without using phenol, the GlassMAX RNA Microisolation Spin Cartridge System is recommended (22).
Total RNA isolated by these methods may contain small amounts of genomic DNA that may subsequently be amplified along with the target cDNA. The presence of this DNA is not likely to cause problems because it lacks the poly(A) region present in the mRNA analyte. As a precaution, however, we recommend performing a control experiment without reverse transcriptase to determine whether a given fragment is of genomic DNA or of cDNA origin. Products generated in the absence of RT are of genomic origin. If your application requires removal of all genomic DNA from your RNA preparation, refer to DNase I Digestion of RNA Preparation.
First Strand cDNA Synthesis from Total RNA
The first strand cDNA synthesis reaction is catalyzed by Invitrogen SuperScript II RT. This enzyme is a mutant of M-MLV RT that has been engineered to reduce RNase H activity, resulting in greater yields and more full-length synthesis (23,24,25). The enzyme exhibits increased thermal stability and may be used at temperatures up to 50°C. In addition, SuperScript II RT is not inhibited significantly by ribosomal and transfer RNA and may be used to synthesize first strand cDNA from a total RNA preparation. The RNA template is removed from the cDNA:RNA hybrid molecule by digestion with RNase H after cDNA synthesis to increase the sensitivity of PCR (26). The AP which primes first strand cDNA synthesis, has been engineered to contain three restriction endonuclease sites and a Not I half-site. Inclusion of these sequences in the primer may facilitate post-amplification cloning using either a restriction endonuclease-based (27) or a T4 DNA polymerase-based (28) method. Because the AP initiates cDNA synthesis at the poly(A) region of the mRNA, it effectively selects for polyadenylated mRNAs; thus, oligo(dT)-selection for poly(A)+ RNA is typically not necessary although incorporating this step may facilitate the detection of rare mRNA transcripts.
Amplification of a Target cDNA
Amplification of a target cDNA requires priming with two oligonucleotides and Taq DNA polymerase. The sense amplification primer is the user-provided GSP,which is specific for the particular gene or sequence of interest and may be designed to include sequence elements that facilitate subsequent cloning steps. The antisense amplification primer is one of the two universal amplification primers provided with the system. The AUAP contains a restriction endonuclease site sequence (adapter region) homologous to the adapter region of the AP. The UAP is composed of the same adapter region plus a dUMP-containing sequence at the 5´ end of the primer required for UDG-mediated cloning. The UAP should not be used to prime DNA synthesis with any archaeobacterial polymerase (e.g., Pyrococcus furiosus, Pyrococcus woesei, etc.) or any long PCR enzyme mixture (e.g., Elongase® Enzyme Mix) that contains one of these enzymes due to the inhibition of polymerase activity by dUMP-containing DNA. Both the AUAP and the UAP will function in PCR at annealing temperatures up to 68°C.e
Design of the Gene-Specific Primer
Efficient and specific PCR amplification is highly dependent on primer design. This is especially true for RACE applications since the PCR is carried out with only a single GSP. In general, effective primers form stable duplexes with their target sequences, are highly specific for their target sequences, and are free of secondary structure such as hairpin loops and dimers (29–31). Additionally, the complementarity of primer 3´-termini must be minimized since primer-dimer artifacts may significantly reduce PCR efficiency. Therefore, dimer formation with the AUAP or UAP primer, as well as itself, should be reduced. Computer algorithms that have been developed (32–35) and are commercially available often facilitate this analysis. Discussion of primer design for RACE applications may be found in Frohman (11) and Loh (6). It should be noted that in cases where only limited peptide sequence information is available, a degenerate GSP may be prepared. The AUAP and UAP included wit the system have been engineered to function at PCR annealing temperatures up to 68°C and to facilitate the cloning step. The user-defined GSPs need to be compatible with the cloning method. Add the following to the 5' end of the GSP: for UDG cloning: 5´–CAU CAU CAU CAU–3´ (use with UAP) for T4 DNA polymerase cloning: 5´–CGA–3´ (use with AUAP)
Nested Amplification
The AP is designed to synthesize first strand cDNA from all polyadenylated mRNAs. The sequence specificity in the amplification reaction is therefore derived solely from the GSP. Often, a second "nested" GSP may be utilized in conjunction with the AUAP or UAP in a second amplification reaction to give the 3´ RACE procedure the specificity of a second primer (9). The nested GSP can anneal immediately adjacent to the first GSP or at sequences within the cDNA further downstream. The nested amplification reaction may be conveniently conducted using a plug of agarose from the gel analysis of the initial 3´ RACE reaction (see Nested Amplification from an Agarose Plug). Ultimately, the 3´ RACE procedure should produce a single, prominent band on an agarose gel. When performing 3´ RACE with a nested primer, sequences specific for subsequent cloning manipulations (see Design of the Gene-Specific Primer) must be designed into the nested GSP.
Cloning of Amplification Products
Conventional cloning methods that typically involve end-repair and blunt-end cloning can be problematic for amplified products (36–38). An alternative is a rapid and efficient method involving the use of UDG (13–16). This method requires that the user design a GSP containing containing a 5'-(CAU)4 sequence. Incorporation of dUMP into the GSP may be accomplished on most automated synthesizers or with Invitrogen Custom Primers (see Design of the Gene-Specific Primer). The product of the 3´ RACE reaction primed with the UAP and the dUMP-containing GSP is treated with UDG, which converts dUMP residues to abasic sites (39,40), to generate 3´ overhangs. The directional nature of the UDG cloning process can be exploited to lend an added level of specificity to the RACE procedure. Only amplification product that results from priming by both the UAP and the appropriately designed GSP are efficient substrates for UDG cloning. Another alternative to conventional cloning methods uses the 3´ to 5´ exonuclease activity of T4 DNA polymerase as the basis for cloning as described by Stoker (28). In this procedure, the AUAP is used in the amplification reaction, and the 3´ RACE products are treated with T4 DNA polymerase to generate a Not I 5´ overhang. Similarly, the user may design a site into the GSP (see Design of the Gene-Specific Primer). Another approach to cloning is to digest the 3´ RACE product using one of the restriction endonuclease sites designed into the AUAP. The user may also design unique restriction sites into the GSP, exploit a site present in the cDNA sequence or end-repair the 3´ RACE product prior to restriction endonuclease digestion (37).
Components are provided in sufficient quantities to perform 20 separate reactions, each converting 1-5 μg of total RNA into first strand cDNA. A control RNA and amplification primers are included in the system to verify performance of the first strand cDNA synthesis reaction and subsequent amplification. Note: The 3´ RACE System does not include Taq DNA polymerase or the reagents required for cloning. Store the 3´ RACE System at –20°C.
|10X PCR buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl]
|500 μl
|25 mM MgCl2
|500 μl
|10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP)
|100 μl
|100 μl
|SuperScript™ II Reverse Transcriptase (RT, 200 units/μl)
|20 μl
|adapter primer (AP, 10 μM)
|20 μl
|universal amplification primer (UAP, 10 μM)
|20 μl
|abridged universal amplification primer (AUAP, 10 μM)
|20 μl
|E. coli RNase H (2 units/μl)
|20 μl
|DEPC-treated water
|1.2 ml
|control RNA (50 ng/μl)
|10 μl
|control gene-specific primer (GSP, 10 μM)
|20 μl
Advance Preparations
Please review the protocols before using this system. You will need the following items not included in the system:
- sterilized 0.5-ml microcentrifuge tubes;
- automatic pipets capable of dispensing 1 to 20 μl and 20 to 200 μl;
- sterilized, RNase-free disposable tips for automatic pipets;
- disposable latex gloves;
- sterilized, distilled water;
- GSP (user-defined, appropriately engineered);
- microcentrifuge capable of generating a relative centrifugal force of 14,000 × g;
- 37°C, 42°C, and 70°C water baths or heat blocks;
- Taq DNA polymerase;
- chloroform;
- mineral oil; and
- thin-walled PCR tubes.
Protocol 1. First Strand cDNA Synthesis
This procedure converts 1 to 5 μg of total RNA into first strand cDNA. Poly(A)+ RNA may be used in this protocol, but is typically not necessary. A control RNA is included in the 3´ RACE System as an aid in verifying that the system performs in your hands. If you decide to use the control RNA as a template for first strand synthesis, simply substitute 2 μl of control RNA (100 ng) in the first strand reaction for your total RNA.
- Mix and quickly centrifuge each component before use.
- Combine 1-5 μg of total RNA or 50 ng of poly(A)+ RNA and DEPC-treated water to a final volume of 11 μl in a 0.5-ml microcentrifuge tube.
- Add 1 μl of the 10 μM AP solution, mix gently, and collect reaction by brief centrifugation.
Note: If you have >5 μg of total RNA, increase reaction volumes and amount of SuperScript II RT proportionately. If you have <1 μg of total RNA, no changes to the protocol are necessary. 50 to 500 ng of poly(A)+ RNA may be substituted for total RNA in this protocol
- Heat the mixture to 70°C for 10 min and chill on ice for at least 1 min. Collect the contents of the tube by brief centrifugation and add the following:
Component Volume (μl) 10X PCR buffer 2 25 mM MgCl2 2 10 mM dNTP mix 1 0.1 M DTT 2
Final composition of the reaction:
20 mM Tris-HCI (pH 8.4 at 22°C)
50 mM KCI
2.5 mM MgCI2
10 mM DTT
500 nM AP
500 μM each dATP, dCTP, dGTP, dTTP
1-5 μg (≤50 ng/μl) of RNA
- Mix gently and collect the reaction by brief centrifugation. Equilibrate the mixture to 42°C for 2 to 5 min.
- Add 1 μl of SuperScript II RT. Incubate the tube in a 42°C water bath or heat block for 50 min.
- Terminate the reaction by incubating at 70°C for 15 min.
- Chill on ice. Collect the reaction by brief centrifugation. Add 1 μl of RNase H to the tube, mix, and incubate for 20 min at 37°C before proceeding to Protocol 2.
- The reaction mixture may be stored at –20°C.
Note: You may stop at the end of step 9.
Protocol 2. Amplification of the Target cDNA
Optimal conditions for amplification are dependent on the nature of each particular primer and target sequence used. Alteration of the magnesium ion, dNTP, or primer concentration, as well as the thermocycling protocol, may be required. The optimal free magnesium concentration for efficient amplification is reported to be between 0.7 to 0.8 mM (29). Since magnesium binds deoxyribonucleoside triphosphates, this factor is affected by both primer and dNTP concentration. In general, lower concentrations of dNTP (50 to 200 μM), MgCl2 (1 to 1.5 mM), and primer (0.1 to 0.2 μM) promote higher fidelity and specificity (41). Higher nucleotide concentration, however, can be used to improve product yield as well as to promote 3´-terminal T-mismatches (42). For a detailed discussion of parameters affecting PCR, please refer to Innis and Gelfand (43) or Saiki (29,30).
The addition of either Taq DNA polymerase, dNTPs, or MgCl2 after reactions have been equilibrated at 75°C to 80°C has been reported to improve the specificity of the reaction (44,45). This "hot start" practice reduces nonspecific binding and the extension of primers during the initial denaturation process. A practical alternative to this classic "hot start" method is to set up reactions on ice then place complete PCR mixtures in a thermal cycler equilibrated to 80°C to 90°C.
- To a fresh 0.5-ml microcentrifuge tube, add the following:
- Add 2 μl from the cDNA synthesis reaction to the tube. Mix gently and layer 75 μl of mineral oil over the reaction. Collect the reaction briefly by centrifugation.
Note: Mineral oil is necessary only when using thermal cyclers that require this barrier.
- Incubate the reaction at 94°C for 3 min. (Extended preamplification denaturation times may impair the efficiency of long (>2 kb) target sequences (46).
- Perform 20 to 35 cycles of PCR, using the protocol accompanying Taq DNA polymerase.
Note: Step 5 is unnecessary if no mineral oil was used in step 2.
- Following amplification, extract the sample with 50 μl of chloroform. Transfer the aqueous layer to a fresh tube.
- Analyze 10 to 20 μl of the amplified sample, using agarose gel electrophoresis and ethidium bromide staining, and the appropriate molecular size standards. If the positive control RNA was used, a single 720-bp band will be visible (see Testing the 3´ RACE System).
Note: If sequences are available for use as internal probes, it is strongly recommended that Southern blot analysis be used to confirm the identity of specific product bands. Specific product can also be identified using a diagnostic restriction endonuclease digestion if the amplified cDNA sequence contains a known restriction site.
Component Volume (μl) 10X PCR buffer 5 25 mM MgCl2 3 autoclaved, distilled water 36.5 10 mM dNTP mix 1 GSP (prepared as 10 μM solution) 1 AUAP (10 μM) or UAP (10 μM) 1 Taq DNA polymerase (2 to 5 units/μl) 0.5
General Suggestions
|Possible Cause
|Suggested Remedy
|No bands after electrophoretic analysis of amplified products
|Procedural error in first strand cDNA synthesis or PCR
|Use the control RNA to verify the efficiency of the first strand reaction (see Testing the 3´ RACE System).
|RNase contamination
|Add the control RNA to the sample to determine if RNase is present in the first strand reaction.
Maintain aseptic conditions to prevent RNase contamination (see Minimizing RNase Contamination).
Use RNase inhibitor during first strand cDNA synthesis.
|Inhibitors of RT present
|Remove inhibitors by ethanol precipitation of the mRNA preparation before the first strand reaction. Include a 70% (v/v) ethanol wash of the mRNA pellet.
Inhibitors of RT include sodium dodecyl sulfate (SDS), EDTA, guanidinium salts, glycerol (>35%), sodium pyrophosphate, and spermidine (18).
Test for the presence of inhibitors by mixing 1 μg of sample RNA ± control RNA and compare yields of first strand cDNA or PCR product.
|Polysaccharide and small RNA (tRNA and 5SRNA) coprecipitation with mRNA
|Ethanol-precipitate the RNA preparation; treat the pellet as described in Lithium Chloride Purification of RNA Preparation (17).
|Target mRNA contains strong transcriptional pauses
|Maintain an elevated temperature after the annealing step and increase the temperature of first strand reaction (up to 50°C) (see Alternative Protocol for First Strand cDNA Synthesis of Transcripts with High GC Content).
|Too much first strand reaction was used in the PCR
|Dilute cDNA reaction 10- to 100-fold. Use no more than 10% of the first strand product in PCR.
|Polymerase from an archaeobacteria was used with dUMP primers
|Use AUAP and non-dUMP GSP in PCR using Elongase® or an archaeobacterial polymerase Use Taq DNA polymerase for PCR with the UAP and dUMP containing GSP.
|Unexpected bands after electrophoretic analysis of "nested" amplification products
|Contamination by genomic DNA
|To test if products were derived from genomic DNA, perform first strand reaction without SuperScript II RT. Pretreat RNA as described in DNase I Digestion of RNA Preparation.
|Spurious priming in the PCR
|Vary the parameters of the PCR according to Taq DNA polymerase instructions and/or use hot start for PCR. Incorporate a preamplification heating step (44,45).
|Poor cloning efficiency
|Inefficient ligation
|Increase the incubation time in the ligation reaction; decrease the temperature.
Ensure the removal of dNTPs prior to ligation by chromatographic separation.
Ensure the removal of vector stuffer fragments by chromatographic separation.
|Poor restriction endonuclease digestion due to residual bound Taq DNA polymerase
|Treat the PCR products with proteinase K (47) (see Proteinase K Treatment of 3´ RACE Products).
|Restriction endonuclease does not digest at the end of molecule
|Add nucleotides to 5´ end of primer. Use a different restriction endonuclease.
The control RNA provided with the 3´ RACE System can be used to troubleshoot both the first strand reaction and the amplification reaction. Use the following protocols, as needed, to troubleshoot for particular problems. To perform th 3´ RACE procedure using the control RNA, you may need the following items, in addition to those listed in Advance Preparations.
- [α-32P]dCTP (3,000 Ci/mmol; 10 mCi/ml)
- glass-fiber filters
- heat lamp
- 10% (w/v) TCA containing 1% (w/v) sodium pyrophosphate
- 95% ethanol
First Strand cDNA Synthesis
- Label two autoclaved 0.5-ml microcentrifuge tubes "A" and "B." Tube A will have an addition of radioisotope t determine the efficiency of first strand synthesis. An aliquot from tube B will be used for amplification.
- Prepare the RNA:primer mixture in a sterile 0.5-ml tube:
- Incubate at 70°C for 10 min and place on ice for 1 min. Collect the contents of each tube by brief centrifugation and add the following to each tube:
- Add 1 μl of [α-32P]dCTP (3,000 Ci/mmol; 10 mCi/ml) to tube A. Final volume will be 20 μl.
- Mix gently and collect the reaction by brief centrifugation.
- Incubate for 42°C for 50 min.
- Terminate both reactions by incubating at 70°C for 15 min. Place on ice for 10 min.
- Collect each reaction by brief centrifugation. Add 1 μl of RNase H to tube B and incubate for 20 min at 37°C. Proceed with tube B to Section Amplification of the Control-Synthesized cDNA.
- Add 80 μl of distilled water to tube A and mix gently.
- Remove two 5-μl aliquots from tube A and spot the aliquots onto glass-fiber filters. Dry one of the filters under a heat lamp or at room temperature. This filter will be used to determine the specific activity of the dCTP reaction.
- Wash the other filter three times in sequence, for 5 min each time, with 50 ml of ice-cold, 10% (w/v) TCA containing 1% (w/v) sodium pyrophosphate. Wash the filter once with 50 ml of 95% ethanol at room temperature for 2 min. Dry the filter under a heat lamp or at room temperature. This filter will be used to determine the yield of first strand cDNA.
- Count both filters in standard scintillant to determine the amount of 32P in the reaction, as well as the amount of 32P that was incorporated.
- Using equation 1, determine the specific activity (SA) of the dCTP in the first strand reaction from the counts obtained from the unwashed filter:
cpm/5 μl
SA (cpm/pmol dCTP) = __________________
500 pmol dCTP/5 μl
- Using equation 2, determine the yield of cDNA from the counts obtained from the washed filter and the specific activity calculated from the unwashed filter:
(cpm) × (100 μl/5 μl) × (4 pmol dNTP/pmol dCTP)
Amount of cDNA (μg) = ________________________________________
The yield calculated for the labeled cDNA in tube A can be assumed as equivalent to that of the unlabeled cDNA in tube B.
- Following first strand cDNA synthesis from the control RNA, you may wish to analyze the remaining cDNA in tube A by alkaline agarose gel electrophoresis or denaturing polyacrylamide gel electrophoresis (PAGE).
Amplification of the Control-Synthesized cDNA
Following first strand cDNA synthesis from the control RNA, the control GSP and the UAP can be used to troubleshoot the amplification reaction. Electrophoretic analysis of DNA products amplified using the control primers should yield a prominent 720-bp band.
- In each of three fresh 0.5-ml microcentrifuge tubes, prepare a PCR mixture as in step 1 of Protocol 2, substituting 1 μl of the control GSP for the user-defined GSP and using the UAP amplification primer. Place tubes on ice.
- In separate 1.5-ml microcentrifuge tubes, prepare serial dilutions of the control cDNA from tube B in sterile, distilled water. Dilute 1:100, 1:1,000, 1:10,000, 1:100,000, and 1:1,000,000. Use the three highest dilutions: control 1: 1:104 (~106 input RNA molecules/μl) control 2: 1:105 (~105 input RNA molecules/μl) control 3: 1:106 (~104 input RNA molecules/μl)
- Add 2 μl from each control dilution to a separate reaction tube from step 1. Mix and layer 75 μl of mineral oil over each reaction. Collect each reaction by brief centrifugation.
- Perform steps 3 through 6 in Protocol 2. Amplify with 30 cycles of 94°C for 45 s, 55°C for 25 s, and 72°C for 3 min. Following agarose gel electrophoresis and ethidium bromide staining, you should be able to visualize a 720-bp band for all three concentrations of input cDNA.
Minimizing RNase Contamination
Successful cDNA synthesis demands an RNase-free environment at all times, which will generally require the same level of care used to maintain aseptic conditions when working with microorganisms. Several additional guidelines should be followed:
- Never assume that anything is RNase-free. Use sterile pipettes, centrifuge tubes, culture tubes, or any similar labware that is explicitly stated to be sterile. Note: Wear latex gloves for all manipulations involving RNA.
- Dedicate a separate set of automatic pipettes for manipulating RNA and the buffers and enzymes used to synthesize cDNA.
- Avoid using any recycled glassware unless it has been specifically rendered RNase-free by rinsing with 0.5 N NaOH followed by copious amounts of sterilized, distilled water. Alternatively, bake glassware at 150°C for 4 h.
- Microcentrifuge tubes can generally be taken from an unopened box, autoclaved, and used for all cDNA work. RNase-free microcentrifuge tubes can be purchased from several suppliers. If it is necessary to decontaminate untreated tubes, soak the tubes overnight in a 0.01% (v/v) aqueous solution of diethylpyrocarbonate (DEPC), rinse with sterilized, distilled water, and autoclave.
- If made with RNase-free labware, most solutions can be made from reagent-grade materials and distilled water, and then autoclaved. Prepare heat-sensitive solutions using sterilized, distilled water, and filtering through a 0.2 μm using sterilized, disposable unit.
- Most aqueous buffer solutions can be treated with 0.01% (v/v) DEPC and autoclaved. Note: Buffers containing primary amines (such as tris) cannot be effectively treated by this method.
- Use aerosol-resistant pipette tips.
Volume (μl)
|Tube A
|Tube B
|control RNA (50 ng/μl)
|AP (10 μM)
|DEPC-treated water
|final volume
Volume (μl)
|Tube A
|Tube B
|10X PCR buffer
|25 mM MgCl2
|10 mM dNTP mix
|SuperScript II RT (200 units/μl)
- Frohman, M.A., Dush, M.K., and Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA 85, 8998.
- Ohara, O., Dorit, R.L., and Gilbert, W. (1989) Proc. Natl. Acad. Sci. USA 86, 5673.
- Loh, E.Y., Elliott, J.F., Cwirla, S., Lanier, L.L., and Davis, M.M. (1989) Science 243, 217.
- Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A., and Arnheim, N. (1985) Science 230, 1350.
- Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Science 239, 487.
- Loh, E.Y. (1991) Methods 2, 11.
- Berchtold, M.W. (1989) Nucleic Acids Res. 17, 453.
- Brow, M.A.D. (1990) PCR Protocols: A Guide to Methods and Applications (Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., eds.) p. 189, Academic Press, San Diego.
- Adams, S.M. and Blakesley, R. (1991) Focus® 13, 56.
- Frohman, M.A. and Martin, G.R. (1989) Techniques 1, 165.
- Frohman, M.A. (1990) PCR Protocols: A Guide to Methods and Applications (Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., eds.) p. 28, Academic Press, San Diego.
- Buckler, A.J., Chang, D.D., Graw, S.L., Brook, D., Haber, D.A., Sharp. P.A., and Housman, D.E. (1991) Proc. Natl. Acad. Sci. USA 88, 4005.
- Nisson, P.E., Rashtchian, A., and Watkins, P.C. (1991) PCR Meth. and Appl. 1, 120.
- Buchman, G.W. and Rashtchian, A. (1992) Focus® 14, 2.
- Buchman, G.W., Schuster, D.M., and Rashtchian, A. (1992) Focus® 14, 41.
- Rashtchian, A., Buchman, G.W., Schuster, D.M., and Berninger, M.S. (1992) Anal. Biochem. 206, 91.
- Sambrook J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
- Gerard, G. F. (1994) Focus® 16, 102.
- Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156.
- Chomczynski, P. (1993) Biotechniques 15, 532.
- Simms, D., Cizdziel, P. E., and Chomczynski, P. (1993) Focus® 15, 99.
- Simms, D. (1993) FOCUS 15, 6.
- Gerard, G.F., D'Alessio, J.M., and Kotewicz, M.L. (1989) Focus® 11, 66.
- D'Alessio, J.M., Gruber, C.E., Cain, C., and Noon, M.C. (1990) Focus® 12, 47.
- Gerard, G. F., Schmidt, B. J., Kotewicz, M. L. and Campbell, J. H. (1992) Focus® 14, 91.
- Hu, A.W., D'Alessio, J.M., Gerard, G.F., and Kullman, J. (1990) Focus® 13, 26.
- Scharf, S.J. (1990) PCR Protocols: A Guide to Methods and Applications (Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., eds.) p. 84, Academic Press, San Diego.
- Stoker, A. W. (1990) Nucleic Acids Res. 18, 4290.
- Saiki, R.K. (1989) PCR Technology:. Principles and Applications for DNA Amplification (Erlich, H.A., ed.) pp 17–22, Stockton Press, New York.
- Saiki, R.K. (1990) PCR Protocols: A Guide to Methods and Applications (Innis,M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J. eds.) pp 13–20, AcademicPress, San Diego.
- Rychlik, W. (1995) Biotechniques 18,84.
- Rychlik, W. and Rhodes, R.E. (1989) Nucleic Acids Res. 17, 8543.
- Rychlik, W., Spencer, W.J., and Rhodes, R.E. (1990) Nucleic Acids Res. 18, 6409.
- Lowe, T., Sharefkin, J., Yang, S.Q., and Diffenbach, C.W. (1990) Nucleic Acids Res. 18, 1757.
- Hiller, L. and Green, P. (1991) PCR Meth. and Appl. 1, 124.
- Craxton, M. (1991) Methods 3, 20.
- Bhat, G.J., Lodes, M.J., Myler, J., and Stuart, K.D. (1991) Nucleic Acids Res. 19, 398.
- Shuldiner, A.R., Scott, L.A., and Roth, J. (1990) Nucleic Acids Res. 18, 1920.
- Duncan, B.K. (1981) in The Enzymes (Boyer, P., ed.), 3rd Ed., Part A, p. 565, Academic Press, New York.
- Friedberg, E.C., Bonura, T., Radany, E.H., and Love, J.D. (1981) in The Enzymes (Boyer, P., ed.), 3rd Ed., Part A, p. 251, Academic Press, New York.
- Eckert, K.A. and Kunkel, T.A. (1991) PCR Meth. and Appl. 1, 17.
- Kwok , S., Kellogg, D.E., McKinney, N., Spasic, D., Goda, L., Levenson, C., and Sninsky, J.J. (1990) Nucleic Acids Res. 18, 999.
- Innis, M.A. and Gelfand, D.H. (1990) PCR Protocols: A Guide to Methods and Applications (Innis, M.A., Gelfand, D. H., Sninsky, J.J., and White, T.J., eds) pp 3–12, Academic Press, San Diego.
- D'Aquila, R.T., Bechtel, L.J., Videler, J.A., Eron, J.J., Gorczyca, P., and Kaplan, J.C. (1991) Nucleic Acids Res. 19, 3749.
- Mullis, K.B. (1991) PCR Meth. and Appl. 1, 1.
- Gustafson, C.E., Alm, R.A., and Trust, T.J. (1993) Gene 123, 241.
- Crowe, J.S., Cooper, H.J., Smith, M.A., Sims, M.J., Parker, D., and Gewert, D. (1991) Nucleic Acids Res. 19, 184.
For Research Use Only. Not for use in diagnostic procedures.


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90 Night Satisfaction Guarantee **
You truly can rest easy on your new mattress from Rocky Mountain Mattress with our 100% guarantee! Your purchase is backed by our 90 Night Satisfaction Guarantee, which gives you a full 90 nights to sleep on and enjoy your mattress in order to ensure that it is the right one for you. Your 90 Night Satisfaction Guarantee begins on the day of delivery.
Sleep on it, roll on it, relax on it – even have someone serve you breakfast in it – and if you decide that the mattress is not for you, returning or exchanging your mattress is easy! Simply repack the product in its original packaging and ship it back to Rocky Mountain Mattress. We will issue a refund of the full purchase price with no re-stocking fees or other hidden charges.
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- All components of the product must be returned in their original condition. Items with tears, stains, punctures, soil spots, hair remains, or any other damage or signs of use will not be accepted. The product must be in the original, new condition in which it was shipped. All components of the product must be returned to Rocky Mountain Mattress within 10 days of the customer receiving a return authorization number.
- The customer is responsible for return shipping, including any and all shipping fees. Please contact us in advance if you plan on returning your mattress, and we will provide complete return instructions and the correct shipping address.
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**The 90 Night Satisfaction Guarantee is not available on adjustable frames, pillows, accessories, and mattress toppers. These items are non-refundable.
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I'm an innovation keynote speaker, author and the Chief Trend Hunter at TrendHunter.com. My keynote speaker bio (at JeremyGutsche.com) has to be a sales pitch to book my keynotes, so this bio is more about me and why I am on this relentless mission to help people Find Better Ideas Faster!
I founded Trend Hunter because deep down, all I ever wanted to be was an entrepreneur, but I could never find my own business idea. Ironically, I built a corporate innovation career helping other people find their business ideas without finding my own. In my final year, I created an innovation pipeline that led my team at a bank to grow a $1 billion portfolio. That sounds great, but it was a striking reminder to me that I still hadn't figured out what I wanted to do.
So, in the wee hours of the morning, I started building Trend Hunter like a giant innovation focus group, an idea pipeline where I hoped people would share business ideas. I also figured somewhere, someone would help inspire me to find my calling. A couple billion pageviews later, and it's clear that serving the Trend Hunter community is my calling!
Today, I see my mission as an important one: to help people find BETTER ideas FASTER, so that they can find their calling and avoid the multi-decade search that kept me hunting ideas for so long. I want to make you BETTER by bringing science to your understanding of chaos and innovation, and I want to make you FASTER by using frameworks and crowdsoucing to help you find the innovation that is right for you.
While starting up Trend Hunter, I also published an innovation book called EXPLOITING CHAOS: How to Spark Innovation During Times of Change. That was 2008/09 and the world certainly went through a lot of chaos. The result was that the book became a bestseller, it landed a couple awards, and I was fortunate enough to be called in by a couple hundred CEOs who wanted me to help them reinvent, faster. It was an incredible experience, both as an innovation keynote speaker, but also as an idea-hungry guy, curious about how innovation works at the world's most powerful companies.
Four hundred keynotes into my journey as a speaker, and I believe I've gained a much better sense of what makes some innovators win while others fail. The experience also helped me prepare for my second book, Better and Faster: The Proven Path to Unstoppable Ideas. I was thrilled to see that book hit the New York Times Bestseller with about 4,000,000 views to the keynote video. That was an incredible feeling for me because this book was much more of a personal journey, including the tales of Trend Hunter, and my unique entrepreneurial upbringing.
On a personal note, I had interviewed my father for the book, because he used to get me to read hundreds of magazines a month, searching for business ideas and brainstorming what projects we would prototype on the weekend. That was obviously a seed for Trend Hunter's inspiration. A week after I interviewed him, he had a heart attack and passed away. So the story, the book, and all the emotion wrapped around that meant that I was particularly proud to see people take interest in the launch. (You can see the innovation keynote videos for both at: JeremyGutsche.com)
When I'm not on the road giving a keynote speech on innovation or chaos, I'm back in Toronto with the kickass team at Trend Hunter.
Basically, I'm an outgoing guy (I suppose any keynote speaker probably says the same) and I love trying to make people laugh. I'm inclined to break rules, adventure and to enjoy a few beers with friends as we chat about ideas, start-ups and my favorite topic: innovation!
I would describe myself as enthusiastic, creative and social. My friends would describe me as hyperactive, short attention spanned and easily distracted. (Mom, this is my attempt at humor.)
A cowboy entrepreneur at heart, I grew up riding dirt bikes and ski racing just outside Calgary, Alberta, home of the Wild West and the legendary Calgary Stampede. I'm the son of a psychotherapist and my entrepreneur father, which explains how I ended up in love with start-ups and creativity. I'm also the younger brother of an art historian. Perhaps I ended up somewhere in the middle of the three. After all, I really like new ventures, meeting new people and artistic creativity.
I've always been obsessed with graphic design, problem solving, and the discovery of innovative new things, which made Trend Hunter my perfect hobby. In the early days, I'd design and code Trend Hunter until 4am while working during the day as a business director at a bank. Today, I design and code Trend Hunter until 4am because I like it.
I'm truly content that pouring my heart into Trend Hunter and my innovation keynotes has resulted in a role where my job and hobby are the same thing.
I'm most in the zone when performing speeches on stage. I fear that many innovation speakers are authors, skilled academics or managers who lack the entertainment fuel needed to spark a revolution. So this is what I aspire to deliver. I like to think of my performances as a mixture of stand-up comedy, audience interaction, big ideas and talking in a really loud voice. I love multimedia keynote presentations and I draw my own animated stick men. Always.
I love meeting Trend Hunters, so if we ever rendezvous, feel free to intrigue me with your random ideas, trends, ideas, creativity and innovation.


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An object thrown on a stone floor in an empty basement. Recorded in stereo with RODE NT4 + ZOOM H4.
Great! This is what I needed. It matches pefectly with scissors driven into a wall :)
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Is Harford County Executive David Craig's plan to give bonuses to local public employees, including teachers, sheriff's deputies and government staff, good public policy?
There's room for disagreement on that question.
Local government employees — including teachers and deputies — have been obliged to make sacrifices in recent years because of the rough economy and financial decisions made by the county's leadership. As a result of those decisions, Harford County government is rather sound and solvent compared to many other counties and to the state and federal governments. The bonuses could be seen as a thanks to the public servants who have taken a hit.
Local taxpayers also have taken an economic hit, however, and there are those who have argued the county surplus from where the bonuses came should be returned to the taxpayers.
Room for argument, indeed.
One point where there is little room for discussion would appear to be on the matter of the union that represents Harford County's teachers, the Harford County Education Association, and the ham-handed, disingenuous way it approached the prospect of receiving unexpected money for its members.
The prudent response would have been to say "thanks" and be done with it. Instead, the union's leadership has fought, unsuccessfully so far, to secure the money in such a way that it would have to be paid through the school system, with no assurance that the money would be distributed as bonus checks to all school system employees.
The HCEA leadership took the position that the bonus money amounted to pay its members were entitled to, claiming a lack of raises in the most recent county budget — a budget drafted in lean times — was a trick to shortchange teachers orchestrated by a county executive, who was once a member of HCEA member and who spent most of his professional life as a teacher and school administrator.
In the back and forth and the gamesmanship the HCEA leadership played with the county executive and the county council, the teachers union leaders forgot to get an answer to one of the most basic questions in negotiating: "What if?" As in, what happens if we hold out for this bonus money to be turned into a salary increase, instead of taking it as a one-time payment? The answer has become painfully clear to school employees represented by HCEA who are "getting nuttin' for Christmas."
Instead of misleading the county executive and the county council about their intentions never to agree to accept the money as a one-time bonus, if the HCEA leadership had been more forthcoming, then 3,200 people would have received $625 holiday bonuses.
The HCEA leadership has taken the position that the reason "Why Bonus Money Must Be Part of Negotiations" is because "Craig is trying to tell the school system how to spend their money. He is not allowed to do this. It is against the law."
It's not the HCEA's responsibility to uphold the law. The union's responsibility, among many things, is to get the most money it can for its members. We're certain that if Craig had sent $100 million to the school system with explicit instructions that it could only be used to double the salary of HCEA members, then the HCEA leadership wouldn't refuse to accept the money because it was against the law for Craig to tell the school system how to spend its money.
It's correct for a union's negotiating strategy to ask for as much as it can, but it's wrong for a union's leadership not to have an alternate strategy for retreat in the face of failure.
The HCEA has a fiduciary responsibility to get the most it can for its stakeholders, not the most it can in the form it wants, but the most money that's on the table. That's another key element of negotiating: Have a pretty good idea of how far the other side will go and say "OK" when that limit is reached.
By being hell-bent on holding out for the bonus money to become permanent pay increases, the HCEA leadership essentially said let its members be damned. That the union leadership ended up walking away with nothing instead of $625 for each person it represents was a bad strategy, one that did not meet its fiduciary obligation.
No matter what transpires next, the HCEA leadership has failed miserably because Christmas will have come and gone and all the county employees, except those represented by the teachers union, had an extra $625 to spend. Perhaps next time, if there is one, the county executive will think twice about offering a bonus that has to be vetted by an organization whose leadership is so adversarial it can neither graciously accept a gift, nor properly represent its membership.


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A fascinating object to indulge in a restorative break at any time. A timeless accessory, the Shot flask for liquor is studied in every detail. It is made of stainless steel with the special Golden Pink finish, Shot is complemented by a funnel that makes filling easy.
The Objets-Bijoux collection colors pink. Small and functional everyday objects, the Objets-Bijoux are characterised by a skillful metal processing. The Golden Pink finish adds elegance and uniqueness to these rich and sophisticated inventions, ideal as a gift to others or to oneself. The different types of objects, selected from Objets-Bijoux, are embellished with this exterior, brilliant and sophisticated finish.
Born in Bergamo, Italy, Laura Polinoro graduated in Drama Art and Music Studies in Bologna. Her first job was in the field of contemporary dance as art director and costume and set designer. Later she undertook research at the Domus Academy in Milan. From 1990 to 1998 she was in charge of the Centro Studi Alessi, where she operated as meta-project designer and art director, coordinating workshops both in-house and in association with various universities and design institutes the world over. She was also involved in publishing and communications activities. Items which entered production in that period are identified with the acronym "CSA". Since 2004 products overseen by Ms. Polinoro have been identified with the letters "LPWK"
He graduated from Politecnico di Milano with full marks in 2005. While studding he started the activity as Interior Designer realizing numerous public and private works. In 2006-2007 he worked for the research center of Baleri Italia, participating to the development of new lines of products. Since the early stages of research his main focus was on product design and on the expressing capacities of the industrial products. Together with the study of Interior projects he realized furniture elements in limited editions. Since 2008 collaborates with LPWK DESIGN.


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The Altura Pocket Rocket is the lightest, least frills waterproof jacket in the Altura range. It packs away really small, weighs next to nothing (110 grams for size medium - without stuff sack - 128g with) and is - surprisingly - actually waterproof.
Features include a (non-waterproof) full-length zip, a full-length stormflap, some reflective trim and detailing and thumb-loops. I'm not sure I see the point of the latter, but they didn't get in the way when not in use, so didn't bother me.
The cut is definitely racy/sparse. Looking around the reviews online, you'll see lots of people commenting on how short is in the body. This is so, but it's something you notice when you're not on your bike. Once you're in a riding position, the shape just works. To give you some idea, the zip is 56cm. On most of my similar waterproofs and gilets, the zip is at least 59cm.
Some of that may be to do with the pocket rocket having a low collar. It does let a bit of wind in, which is a bad thing if you're cold, and a good thing if you need a bit of ventilation. The collar isn't tight at all, giving plenty of space for a buff if it's a bit nippy out.
While the fabric is superbly waterproof, it's not the stretchy stuff (not that you can expect that for this price); there is a bit of breathing space in the chest. I don't mind that at all, it means I can use it throughout the year, it easily fits on top of a jersey and a long sleeve shell. Again to give you an idea, I'm 5'11", 38" chest and weigh 78kg or 12st 4lbs (on a good day) and was testing a Medium. I could probably have gotten away with a Small, but don't feel like the jacket is too big, it's certainly not overly flappy.
There are some reflectives: logo detail on front left collar, logo on front left shoulder, logo on right lower back and reflective stripe across lower back. This is definitely detailing rather than masses of reflectives. If you're going to be relying on a jacket to get seen in the dark, I'd go for the Night Vision Flite we reviewed a little while ago.
The jacket has so far stood up well to daily use. The only thing I've noticed is that the zip snags easily on the storm flap. In my view this is a function of having lightweight fabric - I get this on my other similar jackets and gilets too, but it's worth being aware of. Pulling it too hard may well results in a tear (but hasn't yet).
The only thing that would have made the jacket better (while not compromising on weight/pack size) is a silicone gripper hem around the waist stopping the jacket from riding up.
The stuff sack it comes with is more of a fat sausage shape rather than the round shape you get from others, this makes it quite handy to fit in a jersey pocket.
The Altura Pocket Rocket comes in 6 sizes from XS to XXL, in black or in yellow and can be had for under £50 if you shop around online.
Very good lightweight waterproof at a great price
If you're thinking of buying this product using a cashback deal why not use the road.cc Top Cashback page and get some top cashback while helping to support your favourite independent cycling website
road.cc test report
Make and model: Altura Pocket Rocket Jacket
Size tested: Black/Medium
Tell us what the product is for, and who it's aimed at. What do the manufacturers say about it? How does that compare to your own feelings about it?
Altura say this: "Ultra-light, breathable, waterproof shell with a tiny pack size, the Pocket Rocket offers fantastic low bulk protection from the elements."
Tell us some more about the technical aspects of the product?
* Ultra-light, waterproof and breathable 2.5 layer fabric
* Performance fit
* Tiny pack size (stuff sack included)
* Weight 130g (medium)
* Reflective trim
* 100% Nylon
The jacket seems pretty well put together: taped seams, neat stitching and the reflective logos are showing no signs of peeling off (yet).
Surprisingly waterproof for the lightweight construction, surprisingly breathable for the price!
So far I have no evidence to say that the jacket won't go the distance. Due to the snagging zip and the lightweight material it won't stand up to abuse as a more heavyweight jacket. That's the price you pay for light weight / small pack size...
At 110g for size Medium (a full 20g lighter than the manufacturer's claimed weight) its weight is hardly noticeable.
There are lots of comments floating around on the internet about how it's short in the body. While this is true, you notice it most off the bike - while riding it's not an issue at all - in fact my guess is that it's cut like that so that it doesn't bunch up when you are riding a road bike.
The jacket can be had for less than £50 if you shop around online. That to me is incredible value for money.
Tell us how the product performed overall when used for its designed purpose
It's a great no-frills lightweight waterproof. It does what it says on the tin. Yes, like with all waterproofs, you'll get a bit sweaty if you wear it for a long time, but it's definitely better in this regard than most at this price point.
Tell us what you particularly liked about the product
Its (lack of) weight, its pack size and waterproofness.
Tell us what you particularly disliked about the product
A gripper hem at the waist would have been nice. Other features like pockets, under-arm vents etc would be nice too, but personally I wouldn't choose them at the expense of pack size / weight which is the price you'd have to pay.
Did you enjoy using the product? Yes
Would you consider buying the product? Definitely
Would you recommend the product to a friend? Yes
Anything further to say about the product in conclusion?
At this price point, it's just fantastic value for money. If you're after a no-frills waterproof that's coming with you on every ride and the cut is right for you, look no further.
About the tester
Age: 32 Height: 1.78m Weight: 76kg
I usually ride: All of them! My best bike is: Cervelo Dual
I've been riding for: Over 20 years I ride: Every day I would class myself as: Expert
I regularly do the following types of riding: time trialling, commuting, touring, club rides, fixed/singlespeed, Audax


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Tigers poised to make push for Scherzer extension
DETROIT -- Dave Dombrowski spent most of the past couple weeks focused on finding a closer. The Tigers president/general manager didn't want to discuss what else they might add until he took care of that.
With Joe Nathan now in a Tigers uniform, the closer is on board. The next direction Dombrowski looks for a big contract might be inward.
DETROIT -- Dave Dombrowski spent most of the past couple weeks focused on finding a closer. The Tigers president/general manager didn't want to discuss what else they might add until he took care of that.
With Joe Nathan now in a Tigers uniform, the closer is on board. The next direction Dombrowski looks for a big contract might be inward.
As Dombrowski talked about the benefit of payroll flexibility while introducing Nathan on Wednesday at Comerica Park, he alluded that the flexibility will go in part for their internal concerns.
"A very big part of what we were trying to accomplish [was] to get a closer," Dombrowski said. "And when I say that, sometimes you need some flexibility to make some other things happen, too. We do have some players that are on the verge of being free agents that are pretty big players for us, that you want to create some flexibility there to be in the right spot at various times."
For Max Scherzer, the time is likely this winter or never. For Miguel Cabrera and potentially several others, the time is likely coming soon after. And the more Dombrowski talked about the offseason, the more he alluded to the importance of keeping the core intact.
As he talked about the importance of winning now while maintaining success, he talked about the Atlanta Braves of the 1990s as the model for long-term consistency. Those teams won 14 consecutive division titles.
"One thing you notice with Atlanta, they had a lot of changed pieces," Dombrowski said. "Just look at them throughout the years. But they had [Greg] Maddux and [John] Smoltz and [Tom] Glavine and Chipper Jones. And look at all the other guys they had around them. They had guys that starred for a while, got in there and got out, but they always had the consistent basis of those three starters, year in and year out.
"They had [Steve] Avery who was good and [Kevin] Millwood, and some of them left, some of them became free agents and some of them got traded. But they always had the same main three guys. And so the reality is you can't always have five because then you can't build your club."
The Tigers have two top starters, Justin Verlander and Anibal Sanchez, under long-term contracts. A contract extension for Scherzer would give them that Big Three around which they could build their team.
Nothing is imminent, Dombrowski cautioned, but he expects to approach it at some point soon. He cannot predict the answer, especially given agent Scott Boras' tendency to prefer the open market as the way for pitchers to maximize their contract value, but he sees mutual interest.
"I cannot answer for Scott Boras, but the only thing I would say is I believe Max wants to stay here," Dombrowski said. "When I say that, I know finances and contracts [come up].
"He likes it here. We like him a lot. We'd like to keep him here. Will that lead us to a contract? I don't know. When will those conversations take place? I'm not sure. I know that Scott's got a lot of other free-agent players that he's talking about at this time, but he's a guy that we'd like to keep."
Boras just negotiated a seven-year contract for his top free agent, new Yankees outfielder Jacoby Ellsbury. He has another top free-agent outfielder, Shin-Soo Choo, who has been reported as a top Tigers target. Dombrowski didn't address Choo by name, and he didn't rule out anything, but he downplayed expectations for a major free-agent signing.
"I would think that we would not be involved in the big [names on the free-agent market]," Dombrowski said.
If their next major signing was Scherzer, he would be happy.
"That would be a good sign," he said. "I don't mean to imply that that's imminent either, but I'd be thrilled if Max Schrezer was in a position that he stayed on board with us for an extended time."
Jason Beck is a reporter for MLB.com. Read Beck's Blog and follow him on Twitter @beckjason.


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iRadio: House
iOS Universal Music
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Do you have some trouble? Do you need help? Do you have a awesome idea for this app? Please contact us. We are here to help!
What's New
*** updated servers
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A prolonged militant attack on a naval base in the southern city of Karachi has dealt yet another blow to the image of Pakistan's military.
Militants breached the Mehran navy base in a multipronged assault Sunday. Pakistani security forces regained control of the base, but only after an 18 hour battle that gave attackers time to blow up two reconnaissance planes provided to Pakistan by the US. So far, four attackers and 10 security personnel are confirmed dead.
The Pakistani Taliban said it launched the raid to avenge the killing of Osama bin Laden. Though the Pakistani military had no part in killing Mr. bin Laden the US operation showed Pakistan's military to be helpless to stop the US stealth operation. Many perceived that it put Pakistan in a position of subservience to the Americans.
The naval base attack appears designed to highlight similar weaknesses in the military's capability and remind Pakistanis of the ongoing US relationship by targeting the planes. With few trusted institutions remaining in Pakistan, such attacks on the military undermine the political stability of the country.
"The idea was to shake the state and weaken the institutions and do as much damage as possible to the military and its reputation," says retired Pakistani Gen. Talat Masood in Islamabad. "Coming on the heels of Osama, [the military's] image in the eyes of the Pakistani people is in question."
Air-tight reasons for attack?
Not every analyst is buying this conventional explanation for the attack, however. Some suggest foreign country involvement given the sophistication of the attack and the target.
Describing Mehran naval base as "humongous," military strategist Rifaat Hussain says the militants had to have very detailed maps to find their way to the sensitive P3C Orion aircraft.
Pakistan started receiving upgrades to the P3Cs from the US under a 2006 deal. Four are currently in the US getting upgrades while five were on the Pakistani base before the attack. They are necessary for monitoring Pakistani waters and beyond for ships and submarines – a threat that would come from India's growing naval might, not Islamic militants in the hills.
"It's not possible to maintain [radar] surveillance over the vast [ocean] areas, so you've got these long range aircrafts which carry out maritime surveillance," says P.K. Ghosh, a South Asian maritime expert based in New Delhi. "I think it's a pretty big loss for the Pakistani naval aviation."
Beyond their military use, the planes are also a symbol of US-Pakistani cooperation. Americans employed by Lockheed Martin work on the P3Cs at the base. Six Americans were on the base during the attack but are safe, according to the US embassy.
However, the P3Cs were parked near C-130s, another US-made plane that does play a role supporting missions against Islamic militants, says Mr. Hussain. Given that the strike would benefit India militarily more than the Pakistani Taliban, and given the complexity, Hussain says foreigners may have played a role.
"We need to really figure out who the perpetrators were. To me, it does suggest the involvement of external elements which may have provided them with training and access," says Hussain. "This is beyond the capabilities of the Tehrik-e Taliban Pakistan."
Ambiguity surrounds recent attacks
Pakistan has already seen its fair share of violence this year, with the most recent major attack on a cadet camp in the the northwest Pakistani town of Shabqadir 10 days ago. While the public has grown more immune to bloodshed, Monday's incident appears to have taken on greater proportion, partly due to the fact that it took place in Karachi, Pakistan's largest city, and because a young naval Lieutenant who died has become the "face" of the raids. A facebook page in honor of the photogenic Lt. Syed Yaser Abbas quickly attracted hundreds of followers who posted messages like "You Are The Pride Of Our Country .... You'll Live Forever In Our Hearts ... Insha'Allah."
The ambiguity around who is really responsible for such attacks and just how much the Pakistani military and government works with the United States frustrates the Pakistani public, says Khalid Rahman, a political analyst at the Institute of Policy Studies in Islamabad.
"I think they still trust the military establishment as far as its strength is concerned. But its approach to the whole [war on terrorism] and its dealing with America, that is a major question in their minds," says Mr. Rahman.
For many Pakistanis, he says, the root cause for violence in Pakistan is the spillover of the American war effort in Afghanistan – not terrorist forces from inside Pakistan. Therefore, Pakistanis want to see their leaders take a tough line with the Americans against drone strikes and other military cooperation.
Over the weekend, cricketer-turned-politician Imran Khan led a thousands-strong sit-in designed to block NATO supply routes from Karachi. Mr. Khan's Tehreek-i-Insaaf (Justice Party) has exploited rising anti-American sentiment in the country to call for the removal of the government.
What about Pakistan's nuclear capability?
But for some elites and for many outsiders, the latest attack only raises concerns about the capability of the Pakistani military to keep the country stable and safeguard its nuclear weapons.
"Every such incident weakens the Army's claim of 100 percent nuclear safety," says Pervez Hoodbhoy, a nuclear scientist at Quad-i-Azam University.
"As far as I know, this base did not stock nuclear weapons, but it was heavily defended because there had been repeated attacks on navy personnel in previous weeks. The fact that the Pakistani Taliban could still get through the base defenses suggests that they can get through defenses elsewhere too," he adds.
Issam Ahmed contributed to this article from Islamabad, Pakistan