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By PAUL H. RUBIN
Destin, Fla.
As the oil spill continues and the cleanup lags, we must begin to ask difficult and uncomfortable questions. There does not seem to be much that anyone can do to stop the spill except dig a relief well, not due until August. But the cleanup is a different story. The press and Internet are full of straightforward suggestions for easy ways of improving the cleanup, but the federal government is resisting these remedies.
First, the Environmental Protection Agency can relax restrictions on the amount of oil in discharged water, currently limited to 15 parts per million. In normal times, this rule sensibly controls the amount of pollution that can be added to relatively clean ocean water. But this is not a normal time.
Various skimmers and tankers (some of them very large) are available that could eliminate most of the oil from seawater, discharging the mostly clean water while storing the oil onboard. While this would clean vast amounts of water efficiently, the EPA is unwilling to grant a temporary waiver of its regulations.
Next, the Obama administration can waive the Jones Act, which restricts foreign ships from operating in U.S. coastal waters. Many foreign countries (such as the Netherlands and Belgium) have ships and technologies that would greatly advance the cleanup. So far, the U.S. has refused to waive the restrictions of this law and allow these ships to participate in the effort.
The combination of these two regulations is delaying and may even prevent the world's largest skimmer, the Taiwanese owned "A Whale," from deploying. This 10-story high ship can remove almost as much oil in a day as has been removed in totalroughly 500,000 barrels of oily water per day. The tanker is steaming towards the Gulf, hoping it will receive Coast Guard and EPA approval before it arrives.
In addition, the federal government can free American-based skimmers. Of the 2,000 skimmers in the U.S. (not subject to the Jones Act or other restrictions), only 400 have been sent to the Gulf. Federal barriers have kept the others on stations elsewhere in case of other oil spills, despite the magnitude of the current crisis. The Coast Guard and the EPA issued a joint temporary rule suspending the regulation on June 29more than 70 days after the spill.
The Obama administration can also permit more state and local initiatives. The media endlessly report stories of county and state officials applying federal permits to perform various actions, such as building sand berms around the Louisiana coast. In some cases, they were forbidden from acting. In others there have been extensive delays in obtaining permission.
As the government fails to implement such simple and straightforward remedies, one must ask why.
One possibility is sheer incompetence. Many critics of the president are fond of pointing out that he had no administrative or executive experience before taking office. But the government is full of competent people, and the military and Coast Guard can accomplish an assigned mission. In any case, several remedies require nothing more than getting out of the way.
Another possibility is that the administration places a higher priority on interests other than the fate of the Gulf, such as placating organized labor, which vigorously defends the Jones Act.
Finally there is the most pessimistic explanationthat the oil spill may be viewed as an opportunity, the way White House Chief of Staff Rahm Emanuel said back in February 2009, "You never want a serious crisis to go to waste." Many administration supporters are opposed to offshore oil drilling and are already employing the spill as a tool for achieving other goals. The websites of the Sierra Club, Friends of the Earth and Greenpeace, for example, all feature the oil spill as an argument for forbidding any further offshore drilling or for any use of fossil fuels at all. None mention the Jones Act.
To these organizations and perhaps to some in the administration, the oil spill may be a strategic justification in a larger battle. President Obama has already tried to severely limit drilling in the Gulf, using his Oval Office address on June 16 to demand that we "embrace a clean energy future." In the meantime, how about a cleaner Gulf?
Mr. Rubin, a professor of economics at Emory University, held several senior positions in the federal government in the 1980s. Since 1991 he has spent his summers on the Gulf.
Paul H. Rubin: Why Is the Gulf Cleanup So Slow? - WSJ.com


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May 12: Fujii Rio announces that she has decided to graduate from Kobushi Factory and Hello!Project. Says she wants to focus on becoming a teacher or a nurse. Was meant to graduate at the end of the H!P Summer tour, which ran from July 15 to September 2.
July 6: Up Front announces on the website that they have decided to end Fujii's contract early as a result of noncompliance with the rules. Stated that they had talked with her and her family and tried to resolve the issue first. Magazine reported that it was behavior issues and was having an effect on other members. Fans speculated she wasn't showing up to practice and events because she was absent for a lot of promotional activities for their 4th single.
July 13: H!P website announces that Ogawa Rena has been diagnosed with anxiety neurosis and will go on hiatus until mid-August. Rena posts on her blog telling fans not to worry about her.
July 20: Purikura of Rena with a boy are posted on twitter. Text on photos makes it clear he's a boyfriend. Photos on Kobushi's blog of the exact same outfit make it clear it's her. Up Front makes no statement. Photos in question:
August 4: Photo is leaked of a girl fans believe to be Hamaura Ayano, because of a backpack. Girl isn't able to be clearly seen, but is with a boy and using the charger station on his bike. No statement is released nor is any action taken against Hamaura. Photo in question:
August 16: Up Front states that there is no sign of recovery for Ogawa and that her physical condition is still unstable. Hiatus will continue.
September 6: It's announced that Ogawa Rena has decided to graduate from both Kobushi Factory and Hello!Project. Her final blog post hints that Up Front gave her an ultimatum and she decided to leave on her own. Joins Twitter soon after. Her twitter has recently been locked and is not public. Has also streamed on Line Live and showed Taguchi's face to prove she was the real Rena.
November 9: Photos are leaked on twitter of Taguchi Natsumi in bed with a boy. Photos in question. Up Front makes no statement, Taguchi continues blogging and performing with the group as normal.
December 6: Up Front announces that the contract with Taguchi Natsumi has been terminated. A TV reporter who is a fan of hers says that he knows Up Front Staff and that Taguchi decided to end it herself, had planned to do so at the end of the year but pushed it forward for an unknown reason, and that Ogawa leaving was a big influence on her decision.
December 9: Photos of Hirose Ayaka are leaked on twitter with a man. The twitter account in question is brand new, is writing as if it's from her point of view and is tagging everything #kobushi_bus. Kobushi just started a bus tour.
December 9: Totsugeki Mou Icchou (a show Taguchi has been on before) got word of her leaving and talked about her for a bit. Man from the show named Nakajima sums up what happened and states that the Line Live hurt her more than the actual relationship did. Says she felt guilty, and asked to be withdrawn. Told him to come to the Kobushi Shinjuku ReNy stage or he might regret it, and was not acting her usual self while he was there.
December: someone created a Twitter account under the name Sasuke (さすけ) the other day to leak a purikura of Taguchi and Rena (wearing the same clothes as their infamous LINE LIVE), and talk about "Hirose Hate", "Drinking Factory", "Hirose's ex-boyfriend" and "Wada-Hirose-Taguchi are slutty bitches". Screenshots in question:
Edit by aine: See these two threads for more background.

subset: commoncrawl


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In Luc Besson's sci-fi classic The Fifth Element (1997), the "veryperfect" future human is reverse engineered from a severed hand. As sole surviving fragment of the hijacked Mondoshawan spacecraft carrying the antidote to the "Great Evil," this bronzy limb first appears laid out for examination in a gravel-floored capsule. "A few cells are still alive," analysis reveals, and the hand provides the building blocks for Milla Jovovich's Leelo. Mechanical and chemical processes generate skeleton, tissue, and finally skin... "Reconstruction complete." Initially resembling a prosthetic fused to a new body, the metallic hand has in fact formed a sort of glove around a replacement humanoid limb, which Leelo promptly employs to smash her glass surround—the screen that separates her from the outside world.
Terminating at the wrist, the severed hand of The Fifth Element corresponds to a recent proliferation of hand imagery in art concerned with the framing of human life by digital technologies, as well as the shaping and subversion of these technologies by humans.[1] While the hand is a motif of long standing in art, it plays a crucial new role in this discourse of cyborgian intra-activity, given its ambiguous status as tool and not-tool, human and technology.[2] In this article, we discuss hands as they take on different valences as human and technological actants, performing labor, forging intimacy, and experiencing sensory pleasure. Throughout, we argue that images of the hand in contemporary art problematize the boundary between organic human and inorganic tool, with implications for our understanding of labor, the body, and touch. Where the subject appears impoverished by constant connectivity, several artists break the screen, like Leelo, in a bid to emphasize materiality, the "immediacy and irreducibility of lived experience."[3]


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Last year, T-Mobile announced the LG G2X smartphone at the CTIA 2011. It is basically the T-Mobile's version of the popular LG Optimus 2X. This handset originally runs on the Android 2.2 (Froyo) OS. However, the device was later updated to the Android 2.3.3 (Gingerbread) OS in July 2011. Sadly, the update was pulled, added back and then pulled again.
Now, T-Mobile is rolling out a new update for the LG G2X smartphone. The latest update bumps the LG G2X to Android 2.3.4 (Gingerbread) Operating System. This update will be available automatically via OTA (Over-The-Air) or you can even manually update it via LG's website. It comes with Wi-Fi calling improvements, boot up fixes and more. Check out the complete changelog below.
- Random reboot/power off fixed
- Device stability improvements
- Wi-Fi calling improvements
- Boot up fixes
The latest update is numbered P999-V21y and it can be easily downloaded via OTA (Over-The-Air) until May 14, 2012. After that, you can just head over to the LG's website to manually update your smartphone. The size of the update is just 30.5 MB. If you do not get this update on your smartphone, then just go to Home > Applications > Settings > About Device > System updates and check for the update.
It is advised to backup all you data before updating the device. You should also make sure that your battery is fully charged, before updating the device. If you find any new features after updating your device, then do let us know in the comments section below. To learn more, head over to this page.


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Types of Web Sites
Learn to recognize top-level domains
The one-second technique for getting to know a web site is to look at the URL (the web address) and focus on the top-level domain. That's the part before the first slash, as shown below:
The six most-popular (and original) top-level domains are:
.com = this site is commercial; the intent is to sell
.org = this site claims to be an organization
.edu = this is a recognized educational site
.gov = this is a site sponsored by a government agency
.net = network - kind of a catchall.
.mil = military
But wait - there's more! Lots more -- here are just a few:
.biz = business
.info = information
.name = name
.pro = professional
.museum = museums
... and more
There are also country-level domains:
http://www.handgunlaw.us/ (.us = United States)
http://www.fitness.cz/ (.cz = Czech Republic)
http://www.tripadvisor.fr/ (.fr = France)
http://www.police.uk/ (.uk = United Kingdom)
See the full List of country codes at Webopedia
Creative combinations
You might even see sites combining domains, creating a top-level domain and a secondary-level domain, like this:


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Buy Lexmark W850 toner cartridge at Internet-ink. Get free UK delivery and a 1 year guarantee with all Lexmark W850 toner orders at Internet-ink.
This Lexmark W850 toner is ideal for any small office or busy home that wants to handle high volume tasks and multiple users, at the same time as boosting productivity.
Offering consistency and reliability, the Lexmark W850 toner are a safe choice for personal or professional printing.
The Lexmark W850 toner cartridge delivers a high quality output, producing clear professional texts.
We offer an affordable Lexmark W850 toner cartridge at a fantastic price for your printer, which includes free delivery as standard only from Internet-ink.
You can buy in confidence that you are getting the best value for your money, we compare prices on Lexmark W850 toner and test for quality. This means that all our cartridges are checked and tested before being dispatched without you having to leave the house.
When you buy a replacement Lexmark W850 toner cartridge from us, we give you an all-round service for your printing needs to help get the best value for your cartridge.
By shopping online at Internet-ink you're getting great toner price and service on Lexmark W850 toner cartridges at the top rated online cartridge shop. All our cartridges come with a 1 year guarantee for peace of mind and 100% satisfaction.
Unit 3 Debdale Lane Industrial
Debdale Ln,
Internet-ink is rated 4.8 stars by Reviews.co.uk based on 13250 reviews
(0115) 9371520
Abitech Systems Limited
Registered Number: 4394134
VAT Number: 690343337
Printer and consumable manufacturer's names and logos are registered trademarks of their respective owners. Internet Ink uses cookies to create the most secure and effective website possible.


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WADE CREEK HOUSE ANTIQUES opened in 2007. Located in an old 1940's home, we have expanded many times so there are many areas for you to browse. Located on an acre in Estacada, Oregon, your children can visit with our chickens (yes, we sell eggs) as you look around.
We are much more than an antiques store. We host community fundraisers, have two OUTDOOR VINTAGE MARKETS every summer and hold classes and workshops throughout the year. You can find details BELOW.
OUR VENDORS LOVE TO SHOP FOR YOU. They specialize in old, rusty, antique, primitive, vintage, recycled, revived, repurposed, salvaged, scavenged, chippy, farm, collectibles, indoor and outdoor décor. Come and see what they have found for you!!
SUMMER HOURS April - OctoberMonday - Saturday 10a - 6p and Sunday 11a - 5p
WINTER HOURS - November - March
Monday - Saturday 10a - 5p and SUNDAY 11a - 5p.
Monday - Saturday 10a - 5p and SUNDAY 11a - 5p.
Sunday, June 24, 2018
Plucky and Posh
Subscribe to: Post Comments (Atom)


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"We've had our Versant 3100 for almost one year now and myself and the production staff love it! It is a work horse, but yet produces excellent colors and sharpness. It's a strong and reliable machine, with only minimal service intervention required so far. In summary it is fast, reliable and accurate – it's a 10/10!"
Simon Davidson
Managing Director
Bang On Print & Design Ltd (Newcastle, UK)


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I guess I do things wrong when setup the pchelpware on my win xp. I am thinking to remove the pchelpware.ini to start everything in the beginning, but I don't know where that is. Can anyone give me a hand on that.
I install the pchelpware at C:\Program Files\uvnc bvba\PCHelpWareV2. So what to do?


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Want to know more about this week's guest and topics?
Try clicking on some of these links:
Gen. George Casey, Army Chief of Staff | Bio
Gov. Haley Barbour (R-MS), Chair, Republican Governors Association | Bio
Gov. Ed Rendell (D-PA), Fmr. Chair, Democratic National Committee | Bio
David Brooks, Columnist, New York Times | Bio
E.J. Dionne, Columnist, Washington Post | Bio
Rachel Maddow, Host, MSNBC's The Rachel Maddow Show | Bio
Ed Gillespie, Republican Strategist | Bio
Tom Brokaw, NBC News | Bio
msnbc: Politics
msnbc: Health Care
msnbc: Economy
msnbc: World News - Afghanistan
msnbc: World News - Iran
20th Anniversary of the Fall of the Berlin Wall
David Gregory
Follow David Gregory on Twitter
Follow Meet the Press on Twitter
David Gregory on Facebook
Meet the Press on Facebook


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The Myaamia Foundation has been hard at work over the past year! We'd like to share with you what's new with us.
Since its inception, the Myaamia Foundation was conceptualized as an institution whose sole mission was to help fund activities of the Myaamia Center [www.myaamiacenter.org]. Upon the very generous endowment to the Myaamia Center by the Miami Nation of Oklahoma, we realized that the Foundation was not needed in the same capacity as it once was. We realized that this was an opportunity to reconceptualize our mission to more directly impact and interact with the Myaamia community.
Our new mission is focused solely on becoming a nexus point matching people, skills, ideas, and resources (both knowledge and funding) in order to create language and cultural projects to benefit the Miami people. We do this through awarding grants to projects and by providing a place for people to connect.
For example, say you want to make a children's book based on a Miami story that is written in both Miami and English. Say there is someone else in the community who has always wanted to contribute but didn't know how to do so, but is a fantastic graphic artist. Well, we'd be a hub where the two of you could connect. You could receive cultural and language guidance from the Myaamia Center and maybe you'd receive a grant from the foundation to complete the project.
Or, maybe you want to have a summer youth program for Miami kids, but you need guidance on the curriculum. We could help by connecting you to the right folks and by possibly awarding money to help finance this.
The Myaamia Foundation seeks to further the creation, dispersal, and usage of the knowledge of our history, culture, language, and artistic expression, all the while building and strengthening our community ties.
Please take a moment to take a look at our grant applications and also see what kind of exciting projects we have already funded. Remember, we can't do this good work without you! Please consider donating your time and/or your talent!


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Remember Altar Eagle? The crunchy shoegaze duo who seemed out of place on Type Records with their fuzzy pop debut Mechanical Gardens. Formed by drone king Brad Rose (aka The North Sea) and his wife Eden Hemming, they wrote a completely disarming album, showing how an established artist can still surprise you just by collaborating with the person he shares his fridge with. Three years later and under eerily similar conditions comes a British counterpart: Heterotic, made from Planet Mu head honcho Mike Paradisnas and his wife Lara Rix-Martin. Like Altar Eagle, their focus is overcooked dream pop, so punchy but balletic it could bring John Hughes back from the dead.
Conceived in bed and written on Logic software, there's a intimacy to Love & Devotion, which is more Radio 2 orientated than other Mu fare thanks to some cooing vocals from Warp's Gravenhurst. His sighs over the scissoring keyboards of 'Devotion' have a floaty, abstract quality, the words sounding coming straight from Robert Wyatt's bedside notebook ("May the circle be your problems"). The cold march of 'Blue Lights' finds the singer shrouded in synths and wigging out with paranoia ("Target the ones with the slow reactions/Blacked out windows, no distractions"), while 'Knell' takes ghostly grime sirens and weaves them around slow beats and gentle piano.
Heterotic's passion for synthetic excess means echoing the fun side of the Eighties too, and parts of Love & Devotion are as upbeat as the title suggests. On 'Wartime', chilly keyboards come lit with a delicious squelching bass pad that sounds like someone swallowing frogs, Gravenhurst's groans of "The space between us grows" glazed in dramatic guitar chords. It's as posturing and vivid as adult pop should be, building the mood set by opener 'Bliss' and its Enya synths/squeaky vocals. Paradinas and Rix-Martin have clearly raided their VHS collection to recreate the Reagan-era sound, and 'Robo Corp' is a particular highlight, marching like the score from the similarly-titled movie and featuring an anthemic brass synth borrowed from labelmate Kuedo. The murky children's choir completes the decadent imagery - someone needs to go to Berlin and shoot the video now.
Even when downcast, Love & Devotion is a sensual, lively record and anyone mourning the death of Air France should find much to enjoy here. Just intriguing enough for the Eighties survivor crowd, Gravenhurst's vague but descriptive lyrics recall Karl Hyde from Underworld, conjuring pictures of "Crane flies groping for the glow/Coin arcades and betting shops/Guilty sex and stale cigarettes". As the cool slabs of 'Slumber' melt into snares on 'Fanfare' you'll wish ideas here kept going, pushed deeper into retro-electronica's weirder corners. Here's hoping the sequel includes acid house.
8George Bass's Score


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Cars In Warranty ▼
Cars Outside Warranty ▼
Owners Guides ▼Recycling Book a Service Owners Manuals
Have you visited a Mitsubishi dealership recently? Whether your visit was for Warranty, Recall or any other After Sales work to your vehicle you will be eligible to enter into our prize draw. Enter now for the chance to win a £50 voucher.
* fields ending with an asterisk are mandatory.
Would you like us to let you know about Mitsubishi special offers, events, services and products by email?
Saying yes gives your consent for the details you entered in this form to be used by Mitsubishi Motors in the UK and its affiliates* to send you these emails.
If you change your mind you can withdraw your consent at any time by using the 'unsubscribe' link in the emails we send you or by emailing firstname.lastname@example.com
For more information on the use of personal data by Mitsubishi Motors in the UK, please see our full Data Privacy Statement.
*Mitsubishi Motors in the UK is the trading name of The Colt Car Company Ltd. Its affiliates are Spitalgate Dealer Services Ltd, Shogun Retail Ltd and your dealer Westridge Garage (Ryde) Limited.
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coastal interior design | Tumblr
Coastal Style Interior
Classic Style: Summer beach house interior
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Hi all,
I'm coming up on 2-years postop in March. I am doing well, generally.
However, I still have trouble tolerating the iliac screw on my left side, especially when I exercise.
What does removal entail? Is it a big operation? Are you in the hospital long? Do they remove both, or just the one bothering you?
Also, if you get these anchors removed, do you have a higher risk of fracturing the SI joint later? (Assuming my spine is fused.)
Just not sure it is worth the risks/recovery, but at the same time I would like to be able to exercise without pain.


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The Subject Field
The power behind library catalogs
The subject field is often overlooked and more often misunderstood. The subject field is what makes the library catalog superior to many other information databases. The subject field might also be called Library of Congress Subject Headings, L.C. Subject, Subject(s), Related Subjects, or something similar.
Searching the subject field can provide more accurate results, especially if you're searching a very specific subject or looking for something in a very large library.
Next >>


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Sarah Michelle Gellar is right by her friend Shannen Doherty's side as she battles breast cancer.
In honor of #MondayMuse, Gellar posted a photo of her alongside the former "Beverly Hills, 90210" actress on Instagram and wrote a message about their friendship.
"My #MondayMuse (and really every day #Muse) is my brave friend @theshando. I've always said she is one of the most loyal friends I've ever known, and now while she is facing the toughest journey of her life, she is sharing it with friends she has never met in the hopes of helping other people who are going through the same struggle."
Doherty revealed earlier this summer that she was diagnosed with breast cancer at the same time that she sued her business manager for neglecting to pay her medical insurance.
Last month, the 45-year-old took to Instagram to document her shaving her head after receiving chemotherapy treatment.
U.S. & World
The "Charmed" actress posted six images detailing the heartbreaking process and at the end of the photo series she captioned the final image with two hashtags that truly said it all: #cancersucks and #thankgodforfriends.
Beverly Hills, 90210: Unforgettable Style Moments


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A memorial service celebrating the life of Russ DeVette will be held Friday, Dec. 4, at 11 a.m. at Christ Memorial Church in Holland. Visitation will be Thursday, Dec. 3, from 1-4 p.m. and 7-9 p.m., also at Christ Memorial Church.
Russell (Russ) B. DeVette, teacher and coach at Hope College for four decades, died Monday, Nov. 23, at Hospice House in Holland, Mich. following a long illness. He was 86 years of age.
"Russ DeVette is a man I greatly respect and admire. For many of us who had the privilege of playing for him and coaching with him, he was a father figure of pervasive positive influence," said Hope College president James E. Bultman. "While his athletic and coaching achievements are legendary, I'll always remember him as a man who lived his life with extraordinary Christian convictions. I'll miss him."
DeVette taught and coached at Hope for nearly 40 years, from 1948 until retiring in 1988 as professor emeritus of physical education.
An outstanding basketball player at Hope in the 1940s, he was the first recipient
the most valuable player award from the Michigan Intercollegiate Athletic Association (MIAA).
At times in his Hope career he coached three sports in the same school year. He coached the men's basketball team from 1948-51 and from 1956 to 1977. He was also the head football coach from 1955 to 1969. He remained on the football coaching staff as defensive coordinator through 1987. He also served as head coach in both baseball and women's track. The teams combined to win 14 MIAA championships.
In 2007 the Hope College Alumni H-Club presented him the "Hope for Humanity Award." A holiday men's basketball tournament is named in his honor. The main basketball gymnasium in the college's DeVos Fieldhouse honors him and his wife Doris.
DeVette enrolled at Hope in 1941 from Muskegon as a member of the Class of 1945, but his education was interrupted by service in the U.S. Marine Corps during World War II and he returned and completed his degree in 1947. During his pre-war years as a student he was a member of the 1942-43 "Blitz Kids," the first Hope men's basketball team to go undefeated in MIAA play. In 1947, he was the first player ever to receive the MIAA Most Valuable Player Award in basketball.
After graduating from Hope with degree in history education he went on to the University of Michigan for a master's in physical education before returning to the college to teach and coach.
He served an additional three years in the Marina Corps in the 1950s and coached basketball one year at the Universityn of Maine before returning to Hope.
As a member of the faculty he developed the college's physical education minor, and also served as the athletic director and chairperson of the department. In 1986 he co-authored the book "Coaching Basketball: The Complete Book from Beginning to Championship Play" with faculty colleague Dr. William Vanderbilt, a 1961 Hope graduate who as a student had played for DeVette.
DeVette was head coach of the men's basketball team from 1948 to 1951 and from 1956 to 1977. Over 24 seasons, his basketball teams captured nine MIAA championships and one NCAA regional crown. He was also head football coach from 1955 to 1969, during which period the team won two MIAA titles, and he remained on the football coaching staff as defensive coordinator through 1987. He also served as head coach in both baseball and women's track; his baseball teams won league championships in 1953 and 1954, and his women's track team won league championships in 1981, 1982 and 1988.
DeVette was named the Michigan NAIA Coach of the Year in basketball for 1956-57. From 1975 to 1981, he was a member of the first basketball committee for the NCAA Division III, serving as the committee's chairperson during the last two years. In 1989, he was inducted into the Muskegon Area Sports Hall of Fame.
The college's annual Russ DeVette Holiday Tournament in basketball is named in his honor. During the tournament in December 2005, the main basketball gymnasium in the Richard and Helen DeVos Fieldhouse was named the "DeVette-Van Wieren Gymnasium" in recognition of him and his wife Doris, and current coach Dr. Glenn Van Wieren and Van Wieren's wife Jackie, for their decades-long service to Hope. Glenn Van Wieren, who has been head basketball coach since 1977, is a 1964 Hope graduate and played basketball for DeVette as a Hope student.
DeVette was a member of the Holland City Council for three terms, from 1975 to 1987. His community service through the years has also included coaching and mentoring the Ottawa Hustlers, a basketball team comprised of adults with learning disabilities; Meals on Wheels; Evergreen Day Health; teaching English as a second language with Community Education; The Bridge Store; and serving on the board of Camp Geneva. He was a charter member of Christ Memorial Church of Holland.
Surviving are his wife, Doris, of 60 years; children and grandchildren, Christi and Glenn Hayden of Whitehall (Keith and Jeff), Lynne and David Bouvea of Phoenix, AZ, Joel and Crystal DeVette of Holland (Lisa, Dan, and Heidi), Steven and Shelia DeVette of Grandville (Mike and Brad), Kurt and Jianna DeVette of Holland (Caleigh, Jordanna, and Jarrod), Lisa and Mark Werley of Zeeland (Kirsten, Megan, Ben, Madison, and Braden); brother, Ben and Becky DeVette of Grahm, NC; sisters, Kathy Bremer of Detroit and Marlene Dykstra of Muskegon; sister-in-law, Joyce Lubbers of Tallahassee, FL.
A memorial service will be held Friday, Dec. 4, at 11 a.m. at Christ Memorial Church in Holland. Visitation will be Thursday, Dec. 3, from 1-4 p.m. and 7-9 p.m., also at Christ Memorial Church.


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Davis Mountains State Park to close for major renovations
Starting Sept. 3, Davis Mountains State Park will be temporarily
closed while Texas Parks and Wildlife staff undertakes a major upgrade
of the park's utility system. The closure will last through March 1,
Many of the park's amenities, including the campgrounds, trails, and Interpretive Center, will be unavailable to visitors.
In addition to completing much needed utility projects, an extensive new trail system will be put into place in the Limpia Canyon primitive area. The current system, which consists of 6.5 miles of trail, will be improved and an additional 6 miles will be added to the existing route. The park's new bird viewing area, which began construction in May will be completed, as well.
These improvements will add a new dimension to the park and provide additional opportunities for outdoor recreation for visitors. spring.
Please note that the Indian Lodge and Black Bear Restaurant are unaffected by this closure and will remain open during standard business hours. The Indian Lodge office is open 24 hours a day and can be reached at (432) 426-3254. The Black Bear Restaurant is open Tuesday through Sunday from 7 a.m. to 8 p.m., extending hours until 9 p.m. on Fridays and Saturdays. Advanced reservations for the Indian Lodge can be made by calling (512) 389-8900.


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Station Coordinates - ITRF 2014
The ITRF2014 contributed data files of the 24h VLBI session are formatted in SINEX file format. All files containing datum free normal equation with station coordinates and Earth Orientation Parameter (EOP). To indicate the quality of the contribution, a WRMS is calculated for each or for all stations. The overall WRMS from the session-wise analysis over all stations and for all ACs as well as the combined solution are 3-4mm for the north (violet), 4-6mm for the east (green) and 7-9mm for the height (black) component over all stations, see WRMS graphic. Comparing the individual solution to the combined solution, the combined solution is more precise in evidence. The contributed ITRF2014 results (differences to the mean value) of the station coordinates are shown graphically in X,Y,Z and North, East, Up. The file download open up the combined values of the absolute coordinate and the referred sigma with their mjd of the selected station.


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14 February 2013 | News | By BioSpectrum Bureau
Universal Biosensors reports that its revenue from quarterly service fees from blood glucose test strips increased by 311 percent to $2.2 million
Singapore: Universal Biosensors reported its unaudited financial result for FY 2012 including a doubling of total revenues to $29.6 million for the year. The strong growth in revenue was largely driven by increased sales of the OneTouch Verio blood glucose test strips, which are now being sold globally by LifeScan (a subsidiary of Johnson and Johnson).
In the 12 months ending FY 2012, revenue from quarterly service fees, based on blood glucose test strips sold, increased by 311 percent to $2.2 million, as compared to $544,000 in FY 2011. Product revenues increased by 61 percent from $12.1 million-to-$19.4 million as a result of increased manufacturing volumes. UBI finished the year in a stronger financial position, reporting a net loss of $9.1 million, down 38 percent from $14.7 million in the previous year.
Operating cash burn was reduced by 54 percent (from $7.2 million in the previous year to $3.3 million) and ongoing cost management saw general and administrative costs down a further seven percent on last year, from $7.3 million in FY2011 to $6.8 million in FY2012.
Mr Paul Wright, CEO of Universal Biosensors said, "Strong early growth in sales of OneTouch Verio blood glucose test strips along with prudent management of our operations and overheads saw improved results in 2012. With a solid cash balance and validated technology platforms we are pushing towards the 2013 launch of the first of our coagulation tests developed in partnership with Siemens - an important milestone for the company."
UBI also reported improved manufacturing margins, generating a profit of seven percent from these operations, compared to a loss of two percent in the previous year. UBI has a strong balance sheet with $23.6 million cash and no debt. Mr Wright said the company's improved cash position enabled ongoing investment in its product pipeline. Investment in R&D increased 37 percent, up from $9.8 million in 2011 to $13.5 million. This was partly offset by service revenues of $8 million, received from LifeScan and Siemens for the successful delivery of R&D outcomes. He noted the investment, which is in line with historical levels, was mostly related to the three coagulation testing products in development with Siemens, the first of which is expected to be launched in 2013.


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Be Safe: Indoor Lightning Tips
Do you know what weather phenomenon kills more Americans than hurricanes and tornadoes? It's lightning.
Most lightning deaths and injuries occur when people are caught outdoors during the summer months, mostly in the afternoon and early evening. Lightning seeks the path of least resistance. If you are taller than your surroundings, or are standing next to a tall object (such as a tree), you are a prime target for a lightning strike.
The summer months are the most likely to have more lightning strikes. Although most lightning deaths and injuries occur outdoors, there are risks inside buildings as well.
A safe shelter is a building with electricity and/or plumbing or a hard topped vehicle with windows closed. Picnic shelters, dugouts, small buildings without plumbing or electricity are NOT safe.
Stay off corded phones. You can use cell phones or cordless phones if they are not in a charger. Don't use computers or equipment directly connected to electricity, such as your stove. Stay out of the shower and away from other plumbing as well. You should also stay away from windows and doors. Small cracks in the frames can let lightning in.
Remember Your Pets. Doghouses are not safe shelters. Dogs that are chained to trees or chained to wire runners can easily fall victim to a lightning strike. Bring pets inside.
Protect Your Personal Property. In addition to direct strikes, lightning generates electrical surges that can damage electronic equipment some distance from the actual strike. Typical surge protectors will NOT protect equipment from a lightning strike. If you plan to be away from your home when thunderstorms are possible, unplug unneeded equipment. Do NOT unplug equipment during a thunderstorm as there is a risk you could be struck.
There are three main ways lightning enters structures: a direct strike, through wires or pipes that extend outside the structure, and through the ground. Once in a structure, lightning can travel through the electrical, phone, plumbing, and radio/television reception systems. Lightning can also travel through any metal wires or bars in concrete walls or flooring.
Resource: National Oceanic & Atmospheric Administration


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Compactors in North Lawrence, NY
When you're looking for compactors in North Lawrence, NY, we know that you aren't just searching for the lowest price, but for a reasonable price coupled with great value. Choose Casella to provide your compactors in North Lawrence, NY, and you'll receive competitively priced, competent service that's certain to help make easy your project's timely completion. Contact Casella now to receive a price quote on our compactors in North Lawrence, NY.
We take the guesswork out of pricing, by ensuring that your price quote on compactors in North Lawrence, NY is inclusive. It's a simplified way to get accurate compactors in North Lawrence, NY.
For our business professionals, we are prepared and trained to always bring safe and reliable compactor in North Lawrence, NY that has a low impact on the environment. It's this hands on approach that sets Casella apart from other companies offering compactors in North Lawrence, NY.
Ready to get started? Simply fill out our online form or call us today at 315-842-4067 to find out more.
Casella also offers temporary services ideal for waste collection as a result of construction, remodeling, house and basement clean-outs, special events or any other short-term project. We hold a strong commitment to protecting the environment and converting waste into reusable energy. We're also responsible community members, protecting environmental resources, developing our people, and working to improve where we live and work.
- Customizable container sizes to fit your job.
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Seriously Ken's Rage 1 was one of my favourite games this gen, and will most likely go down as my favourite anime game of all time (sorry Luffy). But 2 was abyssmal, I ad was hugely disappointed about how they removed moves from the first game and simplified combat. It reminded me of how disastrous DW6 was and DW6E. I really hope the franchise doesn't get abandoned and I hope when or if a third game is released we get all the characters having the attention to detail like the first game. I would like to have the meridian system back or something similar and not something terrible like relying on a drop system. Seriously these kind of games should not be about farming stuff, we want to beat crap up and enjoy it.
No clue if anything on here ever reaches the people who can make things happen, but I did suggest One Piece should get the DW treatment wy back here and that actually did happen. Lets see if I can make magic twice, lol.


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Masquerade Wine Dinner at Cafe Modern
- October 31, 2013 6:30 PM
Masquerade Wine Dinner at Cafe Modern
October 31, Halloween Night!
Five-course dinner menu matched with five wines from Bodegas Valentin Bianchi, Argentina
$79 per person (tax and gratuity not included)
Join Café Modern and Chef Dena Peterson to celebrate Halloween Night, modern style! The evening will feature a masquerade reception followed by a spooky themed five-course dinner menu matched perfectly with five wines from Bodegas Valentin Bianchi, Argentina.
Reception Wine: New Age Sangria
Course 1:
Black Lagoon Treat
Thinly sliced stripe bass with truffled ponzu, orange, black peppercorn oil
Paired with Elsa Bianchi Torrontes
Course 2:
Zombie Snacks
Crispy sweet breads with sage butter and autumn root vegetables
Paired with Bianchi Chardonnay
Course 3:
100-Eyed Monster
Veal meatball stroganoff with spiced sweet peas
Paired with Elsa Bianchi Cabernet Sauvignon
Course 4:
Roasted bone marrow and braised beef duo with caramelized onions and crispy baguette
Paired with Bianchi Malbec
Course 5:
Cake of Darkness
Bittersweet chocolate mousse layer cake
Paired with Particular Malbec


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BISC209/F13: Lab5
- 1 LAB 5: Culture Independent Survey of Bacteria in a Soil Community by Illumina™ 16S rRNA gene sequencing
- 2 Background
- 3 Part A: Isolation of Genomic DNA From a Soil Sample
- 4 Part B: Measuring the Concentration of DNA
- 5 PART C: PCR AMPLIFICATION of 16S rRNA gene
- 6 Parts D & E: PCR product "Clean Up" & Agarose Gel Electrophoresis of PCR Product
- 7 Part F: 16S rRNA gene sequencing by Illumina™ Background
- 8 Characterization of Soil Bacteria: Maintaining Pure Cultures of Isolates
- 10 Assignment
- 11 Links to Labs
LAB 5: Culture Independent Survey of Bacteria in a Soil Community by Illumina™ 16S rRNA gene sequencing
In the 1980's scientists discovered that, despite the unicellular character of microorganisms and their invisibility to us, the microbial world is much more diverse and numerous than the macroscopic world of plants and animals. We now know that microbial communities are as complex and interdependent as those of visible forms of life. Traditional measures of diversity relied on physical traits and assessment of microbial metabolism was used for microbial identification rather than for understanding community structure and organization. The use of physical traits in determining relationships between organisms has several major problems when it comes to microbes: 1) microbes are not as morphologically diverse as metazoans; 2) their morphological traits are not indicative of evolutionary relationships; 3) there are no morphological traits common to both macroorganisms and microbes. In the 1980's Carl Woese suggested that the nucleic acid sequences of certain common genes could be used to measure relatedness among radically different organisms. He picked a subunit of ribosomal RNA (rRNA) to serve as the basis for comparison. Ribosomes, the protein-RNA complexes that are the scaffold on which proteins are synthesized, are common to all cells, both prokaryotic and eukaryotic. Despite differences in size, the sequences of rRNA molecules contain regions that are highly conserved, thus highly similar. Woese chose the intermediate sized rRNA molecule, 16S rRNA in prokaryotes and 18S rRNA in eukaryotes, because this gene was large enough to contain enough information for genetic comparisons but small enough for the gene to be sequenced easily. Today, we more commonly use DNA, rather than RNA, for making sequence comparisons and there are other genes that are compared in addition to the 16S rRNA gene. Nevertheless, our largest and most commonly used data base of bacteria today uses the 16S rRNA gene.
Why do we compare DNA sequences in bacteria? Comparing sequences of the gene (16S DNA) that encodes 16S rRNA in different bacteria can be used to identify them with much greater accuracy than traditional identification tools that used a battery of metabolic and physical tests on cultured community members. It is also common now to use rDNA sequencing to deduce phylogenetic relationships between different bacteria and among organisms as diverse as bacteria and humans. Woese's ground-breaking work altered the tree of life and showed that the prokaryotic world was evolutionarily much older than expected and much more important.
Today you will begin a culture independent analysis of the bacteria in your soil community by next-generation, high-throughput 16S ribosomal RNA gene sequencing. This gene encodes ribosomal RNA that is specific to all prokaryotes (bacteria) and is an excellent phylogenetic marker for bacteria. It has been widely used in surveys of microbial community composition and in bacterial identification.
We will use the bacterial primers to amplify the conserved part of the 16S rRNA gene, called 27 Forward (27F) and 1492 Reverse (1492R). The numbers refer to positions on the Escherichia coli 16S rRNA gene. The direction of elongation of new DNA can only proceed in the 5' to 3' direction; therefore, the primer that anneals to base 27 on the 16S rRNA gene template is called the forward primer because DNA synthesis moves downstream on the template. The 1492R primer anneals to base 1492 on the opposite template strand and DNA synthesis moves upstream from the template. Our primers are called "universal" bacterial primers because they anneal to a 16S rRNA region that is highly similar (conserved) to most Bacteria. If we were interested in identifying eukaryotic microbes, such as fungi or protozoa, or if we wanted to identify the Archaea (which are neither Eukarya or Bacteria), we would have to use different primers.
Although our primers have sequences of single stranded DNA that are common to the 16S rRNA gene of most bacteria, the section of the 16S rRNA gene that will be amplified by the DNA polymerase contains highly variable regions of that gene. So variable, in fact, that we should be able to use the DNA sequence of those ~1,500 bases to identify our soil bacteria by comparing the sequences we amplify to the same region of the gene in other bacteria. 16S rRNA gene sequences are archived in an extensive, public database called the Ribosomal DNA Project (RDP) found at | http://rdp.cme.msu.edu/index.jsp;jsessionid=04A058D492CD00120AC01A70AAD7624A . There are so many 16S rRNA gene sequences archived at this site that we should be able to use their powerful, public search engine to identify our soil bacteria down to the genus or species level, if our pcr and the sequencing of the gene from the clones all works well.
Part A: Isolation of Genomic DNA From a Soil Sample
To start the culture independent bacterial identification (sequencing 16S rRNA genes), each student will need a 0.25 gram sample of freshly collected soil. Make sure that your soil isn't too wet to start the isolation. If so, see the Wet Soil option of the Soil DNA isolation protocol below. As a class, assign a single unique identifier code for each student. Use this code as part of the label you will place on each tube containing the various products of the processes we will perform today. At the end of the day, your instructor will freeze 4 tubes from each student that will be labeled as: id code + G (genomic), id code+Ga, id code+PCRp, id code+PCRp+cl
Wet Soil Sample Option
If soil sample is dry or slightly damp you may skip this '"Wet Sample Option'" and start directly with the weighing of an aliquot described below. If soil sample is muddy or really wet (this is unlikely), centrifuge the microfuge tubes with your soil samples at room temperature for 30 seconds at 10,000 x g. Remove as much liquid as possible with a Pasteur pipet before weighing your soil.
Weigh out your aliquots from the freshly collected soil that one of your partners collected before lab. Use the top loading balance and wieghing paper that you are careful not to contaminate. Fold up the edges of the weighing paper to use as a crude funnel and pour the 0.25g of soil into a special PowerBead tube from the MoBIO PowerSoil DNA isolation kit (these tubes will be at the instructor's bench).
Protocol for Using the MO BIO laboratories, Inc. Power® Soil DNA Isolation Kit (cat. # 12888-50).'
Manufacturer Information at | http://www.mobio.com
visual protocol at | http://www.youtube.com/watch?v=v4ggmR1b0pU
Please wear gloves during this protocol
Dry Soil Sample
1. Each student will do the DNA isolation. You will use one 0.25 g soil sample. Add the soil to a PowerBead Tube and label your tube on the top only with your initials and a soil sample identifier code.
What's happening: After your sample has been loaded into the PowerBead Tube, the next step is a homogenization and lysis procedure. The PowerBead Tube contains a buffer that will (a) help disperse the soil particles, (b) begin to dissolve humic acids and (c) protect nucleic acids from degradation.
2. Gently vortex at medium speed to mix.
What's happening: Gentle vortexing mixes the components in the PowerBead Tube and begins to disperse the sample in the PowerBead Solution.
3. Check Solution C1 to see that it's not precipitated. If Solution C1 is precipitated, heat solution to 60°C until the precipitate has dissolved before use. If the solution looks fine add 60 μL of Solution C1 to your PowerBead tube and invert several times or vortex briefly.
What's happening: Solution C1 contains SDS and other disruption agents required for complete cell lysis. In addition to aiding in cell lysis, SDS is an anionic detergent that breaks down fatty acids and lipids associated with the cell membrane of several organisms. If it gets cold, it will form a white precipitate in the bottle. Heating to 60C will dissolve the SDS and will not harm the SDS or the other disruption agents. Solution C1 can be used while it is still warm.
4. Tape your tubes to the Fisher vortex Genie2 so that the tubes lie horizontally.
The tubes need to be securely held by the tape and the tape needs to be attached to the vortex. Allow the tape to be loose enough so that the gasket will vibrate easily but tight enough to be sure the tubes do not fly off while being vortexed. Please wait until at least 2 samples are ready to vortex and then turn it on to full power. One vortex can hold up to 4 tubes (two tubes on top and two on the bottom). Time your samples to "turbovortex" for 10 minutes. Keep an eye on them to be sure they don't "fly away"! Remove tubes at the end of 10 minutes.
What's happening: This step is critical for complete homogenization and cell lysis. Cells are lysed by a combination of chemical agents from steps 1-4 and mechanical shaking introduced at this step. By randomly shaking the beads in the presence of disruption agents, collision of the beads with microbial cells will cause the cells to break open.
5. Microcentrifuge your tubes at 10,000 rcf for 1 minute at room temperature. Caution: Be sure not to exceed 10,000 rcf or the tubes may break. Make sure the PowerBead tubes rotate freely in your centrifuge without rubbing!
6. Transfer the supernatant (don't transfer the beads!) to a clean 2 ml Collection Tube at the instructor's desk. If you don't know what a collection tube is, ask your instructor. Don't use a regular microfuge tube.
Note: Expect between 400 to 500 microliters of supernatant at this step. The exact recovered volume depends on the absorbancy of your starting material and is not critical for the procedure to be effective. The supernatant may be dark in appearance and still contain some soil particles. The presence of carry over soil or a dark color in the mixture is expected in many soil types at this step.
Subsequent steps in the protocol will remove both carry over soil and coloration of the mixture.
7. Add 250 μL of Solution C2 to the collection tube and vortex for 5 seconds. Incubate at 4°C for 5 minutes.
What's happening: Solution C2 contains a patented reagent to precipitate non-DNA organic and inorganic material including humic substances, cell debris, and proteins. It is important to remove contaminating organic and inorganic matter that may reduce DNA purity and inhibit downstream DNA applications.
8. Centrifuge the Collection Tube at room temperature for 1 minute at 10,000 rcf.
9. Avoiding the pellet, transfer up to, but no more, than 600 microliters of supernatant to a new, clean 2 ml Collection Tube.
What's happening: The pellet at this point contains non-DNA organic and inorganic material including humic acid, cell debris, and proteins. For the best DNA yields, and quality, avoid transferring any of the pellet.
10. Add 200 microliters of Solution C3 and vortex briefly. Incubate on ice for 5 minutes.
What's happening: Solution C3 is a second reagent (patented) to precipitate additional non-DNA organic and inorganic material including humic acid, cell debris, and proteins. It is important to remove contaminating organic and inorganic matter that may reduce DNA purity and inhibit downstream DNA applications.
11. Centrifuge the tube at room temperature for 1 minute at 10,000 rcf.
12. Avoiding the pellet, transfer up to, but no more, than 750 µL of supernatant to a new, clean 2 mL Collection Tube.
What's happening: The pellet at this point contains additional non-DNA organic and inorganic material including humic acid, cell debris, and proteins. For the best DNA yields, and quality, avoid transferring any of the pellet.
13. Shake to mix Solution C4 before use. Add 1.2 mL (do this by adding 600 µL twice) of Solution C4 to the supernatant (be careful solution doesn't exceed rim of tube) and vortex for 5 seconds.
What's happening: Solution C4 has a high concentration of salts. Since DNA binds tightly to silica at high salt concentrations, this will adjust the DNA solution salt concentrations to allow binding of DNA, but not non-DNA organic and inorganic material that may still be present at low levels, to the Spin Filters.
14. Load approximately 675µL of the C4 + supernatant mixture from the previous step onto a Spin Filter sitting in a new, clean Collection Tube (save the remainder of the supernatant!!) and centrifuge the spin filter at 10,000 rcf for 1 minute at room temperature. Discard the flow through (NOT the spin filter!!!) and put the spin filter back in the Collection Tube. Add an additional 675 µL of the Step 14 mixture to the same Spin Filter and centrifuge at 10,000 rcf for 1 minute at room temperature. Discard the flow through and load the remainder of the Step 14 mixture onto the Spin Filter in the Collection Tube and centrifuge at 10,000 rcf for 1 minute at room temperature.
Note: A total of three loads for each sample processed are required. You will using the same Spin Filter and Collection Tube for all 3 spins.
What's happening: DNA is selectively bound to the silica membrane in the Spin Filter device in the high salt solution. Contaminants pass through the filter membrane, leaving only DNA bound to the membrane.
15. Add 500 µL of Solution C5 to the Spin Filter in the Collection Tube and centrifuge at room temperature for 30 seconds at 10,000 rcf.
What's happening: Solution C5 is an ethanol based wash solution used to further clean the DNA that is bound to the silica filter membrane in the Spin Filter. This wash solution removes residual salt, humic acid, and other contaminants while allowing the DNA to stay bound to the silica membrane.
16. Discard the flow through (not the Spin Filter) from the 2 mL Collection Tube.
What's happening: This flow through fraction is just non-DNA organic and inorganic waste removed from the silica Spin Filter membrane by the ethanol wash solution.
17. Centrifuge the Spin Filter in the Collection Tube again at room temperature for 1 minute at 10,000 rcf.
What's happening: This second spin removes residual Solution C5 (ethanol wash solution). It is critical to remove all traces of wash solution because the ethanol in Solution C5 can interfere with many downstream DNA applications such as PCR, restriction digests, and gel electrophoresis.
18. Carefully place Spin Filter in a clean 2 mL Collection Tube (provided). DO NOT transfer any liquid that may be on the bottom of the spin filter basket and avoid splashing any Solution C5 onto the Spin Filter.
Note: It is important to avoid any traces of the ethanol based wash solution in the elution that will be created in the next step.
19. Add 35 μL of Solution C6 to the center of the white Spin Filter membrane.
Note: Placing the Solution C6 (sterile elution buffer) in the center of the small white membrane will make sure the entire membrane is wetted. This will result in a more efficient and complete release of the DNA from the silica Spin Filter membrane. As Solution C6 (elution buffer) passes through the silica membrane, DNA that was bound in the presence of high salt is selectively released by Solution C6 (10 mM Tris) which lacks salt.
Alternatively, sterile DNA-Free PCR Grade Water may be used for DNA elution from the silica Spin Filter membrane at this step (MO BIO Catalog# 17000-10). Solution C6 contains no EDTA. If DNA degradation is a concern, Sterile TE may also be used instead of Solution C6 for elution of DNA from the Spin Filter.
20. Centrifuge the Spin Filter in its Collection Tube at room temperature for 30 seconds at 10,000rcf.
21. Discard the Spin Filter. Save the eluent with the DNA. This is your isolated genomic DNA from your soil microbial community. Label the tube with your id code + G
22. Storing DNA: The DNA in the collection tube is now eluted and ready for use as a PCR template after we assess its concentration, keep it on ice. The DNA eluted in Solution C6 (10 mM Tris) must be used immediately or stored at -20 °C to -80 °C to prevent degradation, keep it in your ice bucket for now.
23. Transfer 10 µL of this Genomic DNA to a new, empty color coded for your site (yellow, blue, orange) PCR tube (200 µL) that you will select from the beaker full of empty pcr tubes located on the instructor bench. Label the microfuge tube containing the 10 µL aliquot with your id code + Ga (for aliquot) and bring it to your lab instructor.
24. When you obtain the DNA conc. after using the nanodropper, you will add this conc. information to the labels of both tubes.
25. Make sure your aliquot tube of DNA is labeled and stored on ice before you go with your instructor to the nanodropper to find out the concentration of your isolated genomic DNA. We hope the DNA concentration to be at least 10-30 ng/µL. When you have measured it, add the DNA concentration in (ng/µL) to a class google doc that also has your name and unique id code. Give the aliquot to your instructor to freeze.
Part B: Measuring the Concentration of DNA
The Nanodropper is located in the BISC Equipment room, L308. The NanoDrop ND-1000 Spectrophotometers measure DNA by taking Absorbance at A260nm. This spectrophotometer uses only 1 microliter of sample and does not require a cuvette. The sample is held in place by fiber optic technology and surface tension that holds the sample in place between two optical surfaces that define the pathlength vertically and dynamically. Measurement can be assessed in a range of 2 to 3700 nm/microliter dsDNA. These are expensive machines so make sure you follow the directions carefully and ask your instructor for guidance as needed.
More information is available from the manufacturer's website at: | http://www.nanodrop.com/HowItWorks.aspx
Using the Nanodroppers
1. Clean the upper and lower optical surfaces of the sample retension device by pipetting 2 microliters of clean deionized water onto the lower optical surface. Close the lever arm and tap it a few times to bathe the upper optical surface. Lift the lever arm and wipe off both optical surfaces with a Kimwipe.
2. Open the NanoDrop software from the Desktop of the computer and select the nucleic acids module.
3. Initialize the machine by placing 1 microliter of clean deionized water onto the lower optic surface, lower the lever arm, and select initialize from the NanoDrop software. Once initialization is complete (~10sec.), clean both optical surfaces with a Kimwipe.
4. Perform a blank measurement by loading 1 microliter of highly purified water or Solution 6 (10mM Tris) and select Blank. Note that this blanking step may use something other than Tris, depending on what your sample is dissolve in.
Note that as in traditional spectroscopy, the blank will be subtracted from subsequent measurements. If you want to determine the contribution of a specific buffer or diluent, measure the buffer first using distilled water as a blank. If the buffer does not contribute to the A 260nm reading, then deionized water will be fine to use as the blank. The water or buffer should always be measured to be sure that the instrument has been zeroed properly. The measurement of water or buffer should be zero or very close. All measurements are automatically normalized to 340nm.
5. Measure the nucleic acid sample by loading 1microliter of sample and selecting "measure". Record your DNA concentration. Once the measurement is complete. Clean both optical surfaces with a Kimwipe and the machine is ready for the next sample.
You should ensure that the appropriate constant (50 for dsDNA or 40 for RNA) has been chosen. The software automatically calculates the nucleic acid concentration. If the calculation is done by hand, the A260nm is represented as a 1cm path for convenience, even though 1 nm and 0.2nm paths are actually used during the measurement cycle.
Clean Up
When the last sample in the class has been measured, clean the sampling device by repeating step 1.
PART C: PCR AMPLIFICATION of 16S rRNA gene
If you would like to review the process of DNA amplification by polymerase chain reaction, the link below will take you to the Dolan DNA center's video on the process, | http://www.dnalc.org/resources/animations/pcr.html
Polymerase chain reaction amplification of DNA requires a heat stable DNA polymerase. There are many choices. We will use a proof-reading polymerase called TaKaRa Ex Taq™ from a manufacturer called TaKaRa. Primers, short sequences of DNA that are complementary to a small section of the DNA coding for 16S rDNA, will bind to the template strands extracted from the soil. Primers direct the specificity of the region of the DNA strand to be amplified. The primers that we will use to amplify only the 16S rRNA genes from the bacteria in our soil community are called "Universal" Bacterial Primer sequences. They are "universal" because all eubacteria (prokaryotes) have some form of this gene that is recognized by these two primers. They are named for the base on this gene to which the primer anneals. Our universal primers are 1492R reverse primer TACGG(C/T)TACCTTGTTACGACTT and forward primer 27F AGAGTTTGATC(C/A)TGGCTCAG. Note that the () indicates degeneracy, where either of two bases can be used. Our primers are purchased from Integrated DNA Technologies (IDT).
Setting up the PCR Mix
PROTOCOL WEAR GLOVES AT ALL TIMES AND DON'T TOUCH THE INSIDE OF THE TUBE CAPS OR YOUR PIPET TIPS--Always use a new tip when going into anything in a PCR reaction. (Contamination is a significant problem in PCR)
All reagents for the PCR should be kept on ice and the master mix should be thawed on ice. Since DNA polymerase can function at room temp, we don't want the reaction to start until all the tubes are in the thermal cycler.
The master mix components below have been aliquoted and prepared for you and are in PCR color coded PCR tubes. Note that Master mix contains DNA polymerase, dNPTs, a buffer containing MgCl2, your primers that direct the specificity of the amplification, and nuclease free water (for a total volume of 50 μL). Make sure you label on both the top and sides of the tube
1. Label a PCR tube containing Master Mix with a fine point Sharpie on the top and side of the tube with a your id code and PCRp. The tubes are tiny (holds only 0.2 ml or 200 µL vol) and it is hard to write on them legibly but doing so is very important. Use your fine tipped Sharpie. Make sure the label is legible.
2. Assigned student(s) will set up positive and negative controls for the PCR.
- For the negative control, one person in the lab will add 2µL water in place of the template DNA.
- For the positive control, one person in the lab will add 1 µL of water and 1 µL of E. coli DNA extract.
3. Using a P2 or P10 and special tips with filters, dial 2 μL to be measured. Draw up 2μL of your eluted, genomic DNA (at a concentration between 10-30 ng/µL) into your filter tip and check the tip and be sure you have liquid in it and no air bubbles. This is a tiny volume!
- Remember that the P2 has two red decimal place volume indicators while the P10 only has 1 red decimal place indicator!.
4. Add the DNA to pre-aliquoted 48 µL of Master Mix in a color coded pcr tube. Do this by putting the pipet tip into the master mix and dispensing completely. Before you remove the tip, slowly draw up and dispense fluid a few times to be sure you have gotten all of the template DNA into the tube and remove the pipet tip with your thumb pressed all the way down to the last full stop so that there is nothing in the tip.
5. Mix the reagents in the tube by tapping VERY LIGHTLY on the bottom or "flicking" to be sure that all reagents are mixed and are not adhering to the side of the tube wall.
6. When all the class' pcr tubes are ready, we will pulse (press on&off) the tubes briefly to get all the reagents into the bottom of the tube using a special microcentrifuge rotor.
7. Store your tube(s) on ice until your instructor says it's time to take all the tubes to the thermal cycler. Be sure to wipe off any ice or water on the bottom of the tubes before putting them into the machine.
Component TABLE
|Component & Conc.
|amt. in a 50 µL μl
|Final Conc.
DNAase free
|26.25 μl
|20mg/ml BSA
|2.5 μl
|1 μg/μL
|2.5 mM (each) dNTP mix
|4 μL
|200 μM (each)
|10x Buffer
|5.0 μl
|5 U/μL Ex Taq Polymerase
|0.25 μl
|27F primer
3 μM
|5.0 μl
|300 pM
|1492R primer
3 μM
|5.0 μl
|template DNA
|2 μl
|10-30 ng
- 27F AGA GTT TGA TC(C/A) TGG CTC AG Universal bacterial primer Lane et al. 1991
- 1492R (s) ACG G(C/T)T ACC TTG TTA CGA CTT Universal bacterial primer Lane et al. 1991
The thermal cycler program is, generally, similar for all PCR reactions, but the annealing temperature is dependent on the primer pair. When you design primers, the primer annealing temp. can be calculated based on the GC content and other factors. For 27F and 1492R, a range of 48-55 ˚C is ok, although higher temp. may lead to increased specificity that excludes some organisms' DNA from being amplified.
The length of the fragment you are amplifying determines the extension time. A general rule of thumb is to use an extension time of 1 kb per minute. Here, we amplify with primers designed for the 27th and 1492th positions in the 16S rRNA gene region based on E. coli 16S rRNA gene. Therefore our fragment is expected to be about 1.5kb long, so we will use an extension time of 1.5 minutes per cycle.
Thermal Cycler Program:
|Cycle Step
|# of Cycles
|Initial Denaturation
|3 min.
|30 sec
25-30 sec
2 min
|Final Extension
|10 min
Our PCR program will run for ~2.25 hrs.
Assessing DNA concentration of PCR product:
1. Take the PCR product to the nanodropper to measure the post PCR DNA concentration.
2. Add the DNA concentration info to your PCRp tube label if you can but also add it to the class google doc. Record the conc. in your lab notebook.
BioLabs ladder: a number of proprietary plasmids are digested to completion with appropriate restriction enzymes to yield 10 bands suitable for use as molecular weight standards for agarose gel electrophoresis. The digested DNA includes fragments ranging rom 0.5-10.0 kilobases (kb). The 3.0 kb fragment has increased intensity to serve as a reference band. The 1 kb DNA Ladder was not designed for precise quantification of DNA mass, but can be used for approximating the mass of DNA in comparably intense samples of similar size. The approximate mass of DNA in each of the bands in our 1 kb DNA ladder is as follows:
Parts D & E: PCR product "Clean Up" & Agarose Gel Electrophoresis of PCR Product
ExoSAP-IT® Clean up of your PRC products
It is important that your PCR product be free of left over DNA polymerase, primers, and free nucleotides before a sequencing reaction is attempted on it. There are several methods commonly used to accomplish this purification. One of the simplest is an enzymatic treatment. We will use an enzymatic treatment called ExoSAP-IT® kit from USB. ExoSAP-IT® is a proprietary reagent that treats PCR products ranging in size from less than 100 bp to over 20 kb with no sample loss. The clean-up reagent is proprietary, but it is based on Exonuclease I and Shrimp Alkaline Phosphatase. Recombinant (rSAP) is added directly to the PCR product to degrade primers and to dephosphorylate dNTPs that were not consumed in the reaction.
USB ExoSAP-IT® PCR Product Cleanup (Product Number 78200) NOTE: ExoSAP-IT reagent must be stored in a non-frost free freezer!
1. Transfer 8 µL from your original PCRp product tube to a new empty PCR color coded tube. Label this tube with your id code and PCRp-clean.
2. Add 2 µL of ExoSAP-IT® to the 8 µL of PCR product.
3. Give the smaller vol. of PCR product with ExoSAP-IT® to your instructor to take to the thermal cycler for a two stage process that removes or deactivates contaminants. This part of the pcr product "clean up" takes about half an hour.
The Thermocycler program will perform the following temperature treatments to "clean up" your PCR product so that it will be ready for sequencing.
- A. Incubation at 37 ˚C for 15 minutes to degrade remaining primers and nucleotides.
- B. Incubation at 80 ˚C for 15 min. to inactivate ExoSAP-IT reagent.
SYBR SAFE Agarose Gel Electrophoresis for Electrophoretic Separation and Staining of DNA
To see if you successfully amplified the 16S rRNA gene and not anything else, you will "run a gel" on your PCR products. To run a gel means that we will perform an electrophoretic separation of the DNA fragments in your PCR product, using 1/10 vol. of your PCR product applied to a 1% agarose gel stained with Sybr Safe DNA stain. Your instructor will photograph the gel, label it with your amplicon id from the template and post the gel photo to the Data folder in Resources in Sakai so you can evaluate your success at 16S rRNA gene amplification. You should see a single band of ~1.5kb indicating that the only dsDNA in your PCR product came from amplification of a ~1500 bp gene fragment. Can you explain how we know the size of our amplified gene fragment?
Your agarose gel is made of 1.0% agarose solution (w/v) in 1x TGE buffer (10x=0.25 Tris, 1.9M Glycine, 13mM EDTA) with SybrSafe™ stain.
DNA is uniformly negatively charged and will, therefore, move toward the positive electrode. The separation is determined by the size or mass of the molecule or fragments of DNA.
1. Measure 5 µL of your PCR product using a P10 or P20 micropipet and dispense it as a spot on a small piece of parafilm.
2. Add 5 µLof well-mixed loading dye (0.25% XC, 30% glycerol, 0.1mg/ml RNAase). Mix the loading dye and your PCR product by pipetting up and down. Loading dye contains glycerol to keep our sample in the lane rather than floating away and will have one of 3 marker dyes (bromophenol blue, xylene cyanol, or orange G) that are in the visible spectrum so we can estimate the rate of DNA migration. We don't run our DNA off the gel! (You can't see the DNA since the stain we are using is only visible using UV light.) The gel will "run" at 120V for approximately 30 minutes.
3. Load all of the 10μL mixture of loading dye and PCR product into a lane of a 1% agarose gel (1% wt/vol in TGE buffer (25 mM Tris, 0.19 M glycine, 1.3 mM EDTA) with Sybr Safe DNA stain. Be sure to leave the first two lanes and the last lane empty for the 100 bp ladder, the positive control and the negative water control. (Sybr Safe is a proprietary reagent from Invitrogen that cross-links a fluorescent compound to DNA and allows DNA to be visualized under UV light. It is prepared and used according to the manufacturer (Invitrogen) directions. )
4. Make sure you have recorded in which lane your sample was applied to the provided gel template. Make sure everything is clearly labeled.
5. Make sure you give the rest of your genomic DNA, the rest of the PCR product, and the cleaned up PCR product aliquot (if it is finished) to your instructor to freeze after the gel is loaded.
Imaging of Gel Elecrophesis
1. Your instructor will photograph and label the gel according to the template you have filled out and post the results to the data folder in Resources in Sakai.
How will you judge a successful amplification? How many fragments of DNA and of what size do you expect?
Part F: 16S rRNA gene sequencing by Illumina™ Background
Your instructors will send out your amplicons to a commercial sequencing facility for next-generation, high-throughput DNA sequencing of the 16S rRNA genes found in your soil DNA. We are using Illumina™ sequencing. The Illumina approach relies on attaching fragmented genomic DNA prepared in a sample library to a planar, optically transparent surface on a flow cell. These templates are sequenced using a fourcolor DNA sequencing-by-synthesis technology that employs reversible terminators with removable fluorescence. This highly parallel approach can generate close to 400 billion bases (Gigabases) with high accuracy, with 1.3 billion reads per flow cell run as paired-end 150 basepair reads. Labeled nucleotides are incorporated at each cycle and high sensitivity fluorescence detection is achieved using laser excitation and total internal reflection optics. Images are compiled and processed to produce base sequences for each DNA template. Applications are de novo sequencing where there is no reference available and resequencing, where short sequence reads are aligned against a reference. The genetic differences on the sequences are called using a specially developed data pipeline. We should get up to 15,000 reads per sample! Later in the semester when the data comes back you will have a workshop where we will see the processed data and learn how to evaluate it. The illustrations below come from the Dept. of Energy's Joint Genome Institute (DOE-JGI).
Media: DOE_JGI_Illumina_HiSeq_handout.pdf
Media: illumina_sequencing.pdf
You tube video showing an animated version of the process can be found at [http://www.youtube.com/watch?v=77r5p8IBwJk |http://www.youtube.com/watch?v=77r5p8IBwJk}
Characterization of Soil Bacteria: Maintaining Pure Cultures of Isolates
Continue to subculture your pure isolates to fresh NA plates each week (isolation streak a well isolated colony onto a fresh plate), in the next lab you will make a bacterial smear and do a Gram stain and start other tests to provide examples of co-operative and competitive community behavior among the members of your soil community.
1. All culture plates that you are finished with should be discarded in the big orange autoclave bag near the sink next to the instructor table. Ask your instructor whether or not to save stock cultures and plates with organisms that are provided.
2. Culture plates, stocks, etc. that you are not finished with should be labeled on a piece of your your team color tape. Place the labeled cultures in your lab section's designated area in the incubator, the walk-in cold room, or at room temp. in a labeled rack. If you have a stack of plates, wrap a piece of your team color tape around the whole stack.
3. Remove tape from all liquid cultures in glass tubes. Then place the glass tubes with caps in racks by the sink near the instructor's table. Do not discard the contents of the tubes.
4. Glass slides or disposable glass tubes can be discarded in the glass disposal box.
5. Make sure all contaminated, plastic, disposable, serologic pipets and used contaminated micropipet tips are in the small orange autoclave bag sitting in the plastic container on your bench.
6. If you used the microscope, clean the lenses of the microscope with lens paper, being very careful NOT to get oil residue on any of the objectives other than the oil immersion 100x objective. Move the lowest power objective into the locked viewing position, turn off the light source, wind the power cord, and cover the microscope with its dust cover before replacing the microscope in the cabinet.
7. If you used it, rinse your staining tray and leave it upside down on paper towels next to your sink.
8. Turn off the gas and remove the tube from the nozzle. Place your bunsen burner and tube in your large drawer.
9. Place all your equipment (loop, striker, sharpie, etc) including your microfuge rack, your micropipets and your micropipet tips in your small or large drawer.
10. Move your notebook and lab manual so that you can disinfect your bench thoroughly.
11. Take off your lab coat and store it in the blue cabinet with your microscope.
12. Wash your hands.
Graded assignment:
There is no graded assignment due next lab:) However, there is a graded assignment due at the beginning of Lab 7. If you want to get started on it the instructions can be found at Assignment: Molecular Techniques Theory and Use. Instructions: Assignment: Understanding Molecular Techniques
To Do Before the Next Lab:
You do not need to come in to the lab over fall break but we do want to keep your isolates healthy. Try to start a subculture on fresh NA plates the day you leave for break or the night before and come in to do another subculture the day you return or the morning after. You will need a fresh subculture of each of your isolates in Lab 6. It is important that they be in pure culture. Therefore, don't forget to continue to sub-culture from a single well isolated colony approximately 24-48 hours before Lab 6 (for fast growing organisms) and 3-7 days (based on the growth rate you have observed) for slow growers. Set up a new subculture on a plate using a single, well isolated colony from each of your last streak plates. Please plan ahead to prepare the appropriate cultures so that they will be ready to use when needed. The number of hours it takes from inoculation until a bacterial culture moves from log to stationary or death phase depends on its generation time, the concentration of the inoculum, and other factors.


text:
In the past, if you tried to check out North Korea on Google Maps, you mostly found a lot of blank space. There were some topographical features, such as rivers and lakes, but roads, highways and landmark designations? Sorry.
But on Monday, Google unveiled new, more detailed maps of North Korea. No, Google Chairman Eric Schmidt wasn't tooling around in a Street View car on his recent visit there. Rather, the maps rely on crowdsourced data from volunteers, presumably including people who actually live in that secretive country.
As Senior Product Manager Jayanth Mysore writes in a blog post, this effort has taken a long time:
To build this map, a community of citizen cartographers came together in Google Map Maker to make their contributions such as adding road names and points of interest. This effort has been active in Map Maker for a few years and today the new map of North Korea is ready and now available on Google Maps. As a result, the world can access maps of North Korea that offer much more information and detail than before.
Although you've been able to see satellite views of North Korea for a while, street names and other identifiers have been a mystery. Here's what the area around the capital of Pyongyang looked like before Monday.
And here's what it looks like now, with the new crowdsourced information.
In some instances, there are even a handful of photos linked to specific locations, such as this picture of what's labeled as the Silk Embroidery Institute.
As you'd expect, there's a lot missing. CNN lists a few things you won't be able to find:
The availability of photos quickly thins as users scroll into the North Korean countryside and dries up almost entirely around more controversial areas marked on the map, like the Yongbyon nuclear complex and what Google labels the Yodok and Hwasong gulags.
Human rights groups say as many as 200,000 people may be held in North Korea's network of political prison camps.
The Punggye-ri Nuclear Test Facility, where the regime may be about to carry out a new nuclear test in defiance of international pressure, doesn't appear to be featured on the map at the moment.
Also not available: Street View images. And not all of the crowdsourced information is of high quality. There's this joker, for example, complaining about the quality of fish tacos at the Kumsusan Memorial Palace, which houses the bodies of Kim Jong Il and Kim Il Sung.
You have to admire the courage of anyone in North Korea who helped with the mapping effort. Doing so could land someone in one of those unseen prison camps.
To get a feel for just how bizarre North Korea really is, spend some time reading this blog post from Sophie Schmidt, daughter of Google's chairman, who accompanied him on his recent, controversial trip to North Korea.


text:
When the high-end residential units at the Heights at Groveton open next month, Richmond Highway will begin a new chapter in its long history. A corridor long known for big-box stores and vast parking lots will now feature a new landmark building, one that's already dominating the skyline where Memorial Street crosses the highway. The building has 290 units that rent for $2.75 to $3 a square foot, with studio apartments starting at $1,600 a month.
"These are rental rates you would expect to see in downtown D.C.," said David Ben, marketing director for the Southeast Fairfax Development Corporation. "We've got developers pounding down our doors saying they're willing to go once they see this project does."
A lot is riding on the Beacon of Groveton. As the first-of-its-kind urban development, developer Redbrick Partners is hoping that the market for high-end rental properties will emerge in suburban Groveton. The transformation from urban sprawl to urban scale is one that many people hope will be a sign of things to come along the corridor. Considering the decades of economic studies and planning exercises that have been conducted over the last two decades, some say progress has been sluggish.
"Development and revitalization that has taken place on this corridor has lagged behind," said businessman Kahan Dhillon. "Look at Tysons and Merrifield and Springfield. They've all been revitalizing while we've been writing studies."
ONE VISON OF THE FUTURE includes an expanded Yellow Line, moving south beyond Huntington along Richmond Highway through Mount Vernon. That may seem like a distant dream right now, considering the money and politics caught up in building the Silver Line to Dulles International Airport. But Del. Scott Surovell (D-44) has been pushing the issue for years. Although he acknowledges the discussion would take about 15 or 20 years, Surovell says Richmond Highway offers a realistic opportunity to expand the Metro line south.
"It's not a pipe dream," said Surovell. "This is prime real estate, and if it had proper infrastructure serving it we would have the same kind of development as Arlington and Alexandria and Tysons Corner."
Surovell says that the Richmond Highway corridor offers a perfect storm for Metro expansion. Commercial real estate in Cameron Run leases for $5 more per square foot than in Tysons Corner, an indication that demand can support increased development. Whereas the Silver Line is 20 miles long, extending the Yellow Line to Fort Belvoir would only require about eight miles of track. And because of the recent expansion at the fort, more people work there than the Pentagon. There's even a collapsible wall at the Huntington station.
"It make much more sense to extend a Metro line under that many people than it does to extend it way out into the suburbs like the Silver Line did," said Surovell. "But we don't have the infrastructure, and that's why we need to extend it."