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I have a morning routine that I've refused to stray from in years. At 7 a.m. every morning I brew a cup of strong coffee, grab a blueberry bagel with cream cheese, and start perusing through new and interesting blog content on the Internet. It's the absolute highlight of my day.
Through my years of both writing for blogs and reading them, I've become a bit of an expert on the difference between great and not-so-great blogs. Through my self-study, I've realized that there are only a few vital details that divide the wonderful from the mundane. Sure, anybody can register a domain and start writing their little hearts out, but only a few people can master the art of great blogging. For those of you who are interested in starting a blog or updating one, here are four traits that separate the best from the worst.
Templates and Design
When I designed my first blog, I chose the least cheesy template I could track down. Although it may not seem all that important at first, the aesthetic design of a blog is very important. After all, it is the first thing somebody sees when they visit your blog, so do yourself a favor and put some thought into the appearance of your blog.
Clip art, overly bright colors, awkward backgrounds, and overbearing typography don't look good in design, so try and stick with something that's clean, not too flashy, and creative. You can purchase templates, design your own, or search amongst free ones. No matter what you do though, just go with something that stands out.
Spelling, Grammar, and Punctuation
For the life of me, I can never figure out why people allow themselves to make mistakes on their blogs. If you're writing for a blog, there is no reason you should let errors slip through the cracks.Treat your blog like a school project and read through all your posts before you publish them to make sure they are clean.
Sure, people may forgive one or two errors along the way, but can you imagine how unprofessional you appear when you overlook things like the difference between you're and your? If you want your blog to be respected, make sure to treat it with respect and edit your work.
Most of the time it isn't the content of a blog that attracts readers, it's the author. As the writer of a blog, it is your duty to bring some personality to the posts you write. Tell your audience a bit about yourself: who you are, what you do, and why it is you write a blog.
Once the audience gets to know you a bit, they'll be more attached to your blog's content. Furthermore, if you are writing something like a food blog or movie blog, tell readers why you are an authority on those things. Perhaps you work as a head chef in a restaurant or you interned with Steven Spielberg; either way, always try to bring your personality to the table when you write posts.
Daily Updates
Have you ever come across a riveting blog but then noticed it hadn't been updated in months? There are blogs out there that have a lot of potential, but many of them go un-updated for weeks or months at the time. Not only does doing something like this make you look like you don't care about your blog, it makes you look apathetic, which is a great way to lose followers.
If you've started a blog, make it a routine to update the content at least once a day. It might be a tough commitment to stick to at first, but keeping your blog updated will help you gain followers and build up your blog's prestige.
Final Words…
There are millions of blogs out there on the web, but many of them aren't what they should be. If you are looking to create a respected, popular blog, take into consideration making daily updates, editing your posts, bringing a personality to your writing, and utilizing a creative design template. By doing those simple tasks, you'll be on your way to creating an impressive blog in no time!


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Easy, affordable ways to make and keep your favorite memories.
Homespun Memories for the Heart offers more than 200 inventive ideas and examples to help you make unforgettable moments, from growing sunflower playhouses to creating alphabet prayers and garden parties. You'll find:
- inventive gift ideas
- mouth-watering recipes people will want to gather around
- creative ways to observe holidays
- imaginative crafts for snow days and sleepovers
Plus, learn how to celebrate "just because" events like rainy days, road trips, bedtime, and book-reading--because even ordinary moments are enough reason to celebrate.
Publishers Description
Beyond birthday parties, anniversaries, holidays, and holy days, there is the every day. And while the loss of the first tooth or the first day of school may seem like normal, ordinary occurrences, these authors show that even "just because" events are enough reason to celebrate the One who gives us life each day.
There is spirituality behind all celebration. With more than 200 inventive ideas plus Christian inspiration, brief reflections, and biblical examples of everyday celebrations, the authors give concrete, practical, and tangible ways to bring that spirituality into everyday. Readers will be encouraged to create their own family traditions and bring faith to life by making home a place where Christ, family, and friendships can be celebrated openly and often-because life doesn't have to be ordinary.
Buy Homespun Memories for the Heart: More Than 200 Ideas to Make Unforgettable Moments by Karen Ehman, Kelly Hovermale & Trish Smith from our Christian Books store - isbn: 9780800759834 & 0800759834
The team at Christian Bookstore .Net welcome you to our Christian Book store! We offer the best selections of Christian Books, Bibles, Christian Music, Inspirational Jewelry and Clothing, Homeschool curriculum, and Church Supplies. We encourage you to purchase your copy of Homespun Memories for the Heart: More Than 200 Ideas to Make Unforgettable Moments by Karen Ehman, Kelly Hovermale & Trish Smith today - and if you are for any reason not happy, you have 30 days to return it. Please contact us at 1-877-205-6402 if you have any questions.
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Wants to Meet Up
- Last login 5 months ago
Join Couchsurfing to see Lu's full profile. It's free!
About Me
Get out of comfort zone, learn more, and explore more
I love to embrace all new chances and adventures. I believe that to know more about myself and the world,I have to venture outside so my current goal is to travel as much as possible.I'm easy-going,enthusiastic and always trying to be helpful ;) I love to talk and share my experiences and knowledge.
I walk slowly, but never walk backwards.
Misfortune may be a blessing in disguise.
Why I'm on Couchsurfing
I heard it from my roommate and found it fantastic!
I "fled" from a village in Sri lanka and one of the hosts here kindly offered me a safe place to stay and calmed me down. And without the help from two guys from Male in Maldives, I don't believe I could have such a nice experience there. Many experiences in the Middle East and Africa.
Read and get inspired, listen to beautiful music, learn new languages and above all, travel and know different people!
- chinese new year
- traveling
- music
- languages
Music, Movies, and Books
the Vampire Diaries
jazz, rock n roll..
One Amazing Thing I've Done
It took me 17 consecutive hours to reach the peak of the sacred mountain in Sri Lanka, Adam's Peak, with all the hardships such as the keep-emerging leeches,rain, wet road, darkeness, and cold river..on the day of Chinese New Year! I also have independently taken my mom to 7 countries in South Asia to travel.
Teach, Learn, Share
I can teach Chinese, show how to use chopsticks, tell how to take good photos, and I have a lot of travel experiences to share
What I Can Share with Hosts
Stories, viewpoint, skills
Countries I've Visited
Brunei Darussalam, Cambodia, China, Cuba, Egypt, Georgia, Hong Kong, India, Indonesia, Iran, Israel, Japan, Jordan, Kenya, Laos, Macao, Malaysia, Maldives, Mexico, Myanmar, Nepal, Palestine, Philippines, Russian Federation, Rwanda, Singapore, South Korea, Sri Lanka, Thailand, Turkey, Uganda, United Arab Emirates, United States, Viet Nam
Countries I've Lived In


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The Crisis Bread Basket needs you to make a difference:
- We Need You – please contact firstname.lastname@example.com to get more information about volunteering or potential internships.
- Set up a food drive or donate food.
- Refer someone in need. If you know someone in need – please feel free to contact us or tell them to stop by during distribution hours.
- Donate money.
- Spread the word. English Info Card. Spanish Info Card.


text:
From the sourcing of raw material till the final dispatch, our quality auditors keep a stern eye on every sage. Supported by a well-organized network and dependable resources, we have developed as a reputed supplier of automobile cylinder.
Sir Vinayaka Enterprises
Plot No 116 1 Auto Comlex Sagar Road Shimoga - 577204 Karnataka India, Karnataka , India
Owing to our well-maintained and organized inventory system, we deliver the same in the committed time. We are reckoned as the noted Supplier of automobile cylinder as we are capable of meeting the exact requirements of the buyers.
Vinayak Automobile
Tangandadi Road , Muddebihal, Muddebihal, Bijapur, Karnataka , India
It is due to our guarantee of providing damage-free products to the clients; we are highly in demand amongst the buyers. Our packaging experts keep a stringent check on the entire packing procedure ensuring the same. We follow strict quality procedures that are as per the guidelines, help us in presenting dependable and cost-effective product range.
Kalyani Agency
No. 516, Konappana Agrahara, Electronicity Post, Hosur Main Road, Next To Infosys, Bengaluru, Karnataka - 560100, India
Product Description : In order to cater the diversified demands of our clients, we have been able to provide a unique range of Automotive Hydraulic Cylinder. Features : Impeccable performance Easy to use Flawless operations Other details : The primary emphasis of the Cylinder is on the mid-duty application range of vehicles. The cylinders have a Bore range of 40 to 150mm with the limit being 300mm, a stroke range of
Belgaum Hydropower
Khadarwadi Cross, Om Industrial Estate, Rajaram Nagar, Udyambag, Karnataka - 590008, India
As we believe in client's supremacy, we decide the acceptable payment methods at the time of undertaking the agreement/contract. Moreover, our payment processing methods are secured. To handle both automated inbound and outbound orders, we have exceptional warehousing solutions. We keep the wide array of products sorted in the warehouse to make sure that the delivery commences systematically.
Veerganpati Autogas
Deshapande Nagar, Hubli, Karnataka - 580029, India
Deals in Industrial Cylinder, automobile cylinder
Varalakshmi Machines Pvt. Ltd.
C 92 Peenya Industrial Estate Peenya 3rd Stage, Karnataka - 560058, India
Deals in Automobile Components, crankcases, Insert Crank Case-LH, Flywheels, wheel bearing retainer, Automotive Sleeves, Break Drum, Center Clutch, motorcycle center clutch, Brake Drum, Flywheel-02, Flywheel-01
C-2, Govt. Indl. Estate, Udyambag, Karnataka - 590008, India


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Judge Punts On Apple's Bid To Bar Samsung SEP Claims
In her first order on one of the numerous post-trial motions filed by both sides, Judge Lucy Koh said that ruling on Apple's claims of misconduct by Samsung would not provide it with any meaningful relief, since the jury ruled in August that Apple did not infringe Samsung's patents. ...
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This specific word was released to a Scribal Company by Apostle Bernard Boulton.
"The children have come to give birth, but there is no strength to bring them forth." Isaiah 37:3
The Lord says that My Scribes are in a birthing season. They are birthing creativity from their dreams and visions. They are birthing from their desires the things that I have deposited and designed in their hearts.
My Scribes must have strength to bring forth what they carry. They must break out and break forth for I am giving them strength to bring it forth. I am giving them wisdom to know what to birth in what seasons for they must know my perfect timing.
The strength to bring forth what they carry is my dunamis. My Scribes must declare Acts 1:8 "And you shall receive power (Dunamis) when Holy Spirit has come upon you and you will be my witnesses in Jerusalem, Judea, Samaria and to the uttermost parts of the earth." My Spirit is coming on My Scribes to give them power to carry and bring forth what they are carrying.
I am making you strong to give birth. I am breathing on you new energy to bring forth what you are carrying.
My Scribes will not be like Rachel who died giving birth to Benjamin because she had no strength to bring him forth. I am calling you to life and not death. I am imparting strength to you to bring forth Benjamin (Genesis 35:16-19).


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I 'm Trying to Move On and Better My Self
Personal Stories, Advice, and Support 53 People
Still At Mom's House And UmemployedI graduated two years ago from college. I finished my studies in December, but due to an incomplete paper did not officially graduate until August of 2009. I was trying to hold on to the community I had joined for 6 years (college took that long for me). I ended up causing...
LitamalianTherpo 31-35, M 2 Responses 1 Jan 26, 2011
Cant Move Onhi why cant i move on from my girlfriend i miss my kids and i am making my mum and dads life a nightmare all i seem to do is drink and gamble and mope about
shocklance 36-40 1 Response 0 Jul 4, 2010
How Can I Let Go Of False Hopes?I won't call them dreams anymore. I won't waste anyone's time with unneccessary details but I finally realize that all these unrealistic aspirations were really just coping mechanisms for me, an escape, a belief that I could somehow rise above and escape a "real world" lifestyle...
SCCK441 18-21 1 Response 0 Jun 19, 2010


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tag:blogger.com,1999:blog-8150928949507396757.post1347266099831330367..comments2018-01-06T03:47:20.465-08:00Comments on I Don't Want To Turn 50 Like This : The light that came from nowhere.Giving Up Drugs and Alcoholhttp://firstname.lastname@example.com:blogger.com,1999:blog-8150928949507396757.post-15755846027033276002017-02-17T12:08:34.720-08:002017-02-17T12:08:34.720-08:00The possibilities are endless. The possibilities are endless. Anne Ainsobrietyhttps://email@example.com:blogger.com,1999:blog-8150928949507396757.post-13823607941771558252017-02-17T12:07:27.186-08:002017-02-17T12:07:27.186-08:00I remember day when the black clouds parted and th...I remember day when the black clouds parted and the sun peaked through. When I realized that sobriety might just change everything.
There is so much light and love. It isn't always easy to find, but it is there.

Keep going. See what happens.
AnneAnne Ainsobrietyhttps://email@example.com:blogger.com,1999:blog-8150928949507396757.post-57593004327349165772017-02-15T22:54:32.054-08:002017-02-15T22:54:32.054-08:00I have been reading over your 100 days back in Aug...I have been reading over your 100 days back in Aug last year and then to October and it's crazy how obvious it is the struggle with alcohol we have in our posts.
It is kind of like the alcohol demon takes all of our brain matter and scrambles it then changes it to make us think that we-have-a-problem-but-we-blame-everything-else-first type of thing.
then without alcohol the freedom in your writing. I wish i was doing so Well but it does come and go a lot from this end.
Congrats on the 100 days to you too xxx MxxGiving Up Drugs and Alcoholhttps://firstname.lastname@example.com:blogger.com,1999:blog-8150928949507396757.post-82990704345013508862017-02-15T22:47:37.989-08:002017-02-15T22:47:37.989-08:00It's funny Wendy, today out of the blue I was ...It's funny Wendy, today out of the blue I was in the super market and thought "wouldn't it be great to have a glass of wine tonight"
After kids basketball, cello picking up dropping off - it is so disjointed. Working is soooooo much easier and facing this mundane taxi service (with no money at the end!) is a pain in the neck.
But it is good, and it is right - and it is my responsibility. So I am going to keep going. It will get easier.
M xxGiving Up Drugs and Alcoholhttps://firstname.lastname@example.com:blogger.com,1999:blog-8150928949507396757.post-63847050319218159672017-02-15T20:00:19.398-08:002017-02-15T20:00:19.398-08:00100 Days Today! I am sorry to hear about the hard ...100 Days Today!
I am sorry to hear about the hard times you had with your ex, but glad you have a good relationship today.
Growing up is hard.
There are certain areas in my life I still have a ways to grow.
What I love about this post, is the light and joy you are seeing today, is real, and it will continue to bloom!
xo
Wendy

Un-Tipsy Teacherhttps://firstname.lastname@example.com:blogger.com,1999:blog-8150928949507396757.post-63815082933184876452017-02-15T03:33:42.544-08:002017-02-15T03:33:42.544-08:00I love this so much. I personally believe I can on...I love this so much. I personally believe I can only feel joy when I don't drink. The feeling never comes while drinking, it's numbed all the time. But every time I'm sober here it is. I can walk down the street and it hits me, driving in my car listening to music and it hits me. I love it. Also genuine excitement is a feeling I get now too which I don't while drinking.

As for not having a clue what you were doing with your life, you are not alone. I shudder to think of some of the stuff I have done, my entire 20's were all over the place. I think back to that person and wonder who the hell it was.

I'm so happy for you, I truly believe that we are just at the beginning of our journey at day 100 so can't wait to hear what happens next! XPutting Down The Glasshttps://firstname.lastname@example.com


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Dr. Salvi was recently interviewed by the Top of Mind radio show, which can be found here. The article under discussion can be found here.
A study by the Center for Speech and Hearing, which explores new behavioral tests for diagnosing inner ear damage, has been the focus of an article in UB News. You can find the article here.


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MILAN, Feb 16 (Reuters) - Italian food company Barilla is to spend around 50 million euros ($53 million) on increasing production of pasta sauces to meet rising demand from consumers, the company said on Thursday.
Under the investment plan the company intends to expand the group's dedicated plant near Parma, in northern Italy, making it the largest producer of pasta sauces in Europe, with up to 122,000 tonnes a year.
Once completed, the expansion will enable the plant to produce meat-based sauces in addition to the existing range of pesto, vegetable and tomato-based sauces.
The family-owned group, which is the world's biggest pasta producer and third largest maker of sauces, opened the plant in 2012 and has since increased its sales of sauces by 45 percent. ($1=0.9381 euros) (Reporting by Francesca Landini; Editing by Greg Mahlich)


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Zuku gives you ease of managing your account on the go through you mobile phone.
Ensure you have downloaded and installed the Telegram Messenger app which is available for free on Google Play Store, Apple Store, and Windows Store. Enter your phone.
Once installed, type "ZukuFiber" in the search bar at the top right of your screen.
Select ZukuFiberBot
Click on start / (restart) to display options Menu
Select Last My Zuku Bill from the options menu, Enter your account number and wait for your details to be processed.
Tell us where you think George-The-Goat is!
Fill in the form below to get our customer care team to get in touch with you


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Every week, I document another dish that impressed and satiated me during my food adventures around New York City
I have been wanting to come out to try Lali Restaurant for quite a while now. But the little Dominican counter is located pretty far west in Hell's Kitchen and is only open for lunch. The odds of me being in this neighborhood at this time have been against me since I first heard about them over a year ago.
But fate finally struck. I had a special private tour that started at a Hell's Kitchen hotel in the mid-afternoon. So I knew exactly where I was going to get lunch.
Lali is about as authentic as you get. There are a few seats in the back of the restaurant, but it's mostly a counter surrounding a little kitchen where two women dish up homecooked Dominican plates. The language heard here is Spanish more than English and while the sweet woman spoke English, I got to practice my broken Spanish which is always exciting.
The menu is full of rotating specials and one of their most popular was available on the Monday I arrived – a chicken stew known as pollo guisado.
This is about as hearty and comforting as lunch can get. Two pieces of tender juicy chicken were perched on a generous pile of yellow rice and beans. Chunks of the chicken fell off the bone and mixed in with the other ingredients. It had just a hint of spice with lots of herbs and garlic. Most importantly, I didn't need to eat again for many hours.
There is so much to eat in this city and while I try to get everywhere, some places – like Lali – are more difficult than others with my schedule. But with a little patience (and fate), I'll get to eat as much delicious food as possible. Price: $9
|630 Tenth Avenue (between West 44th and West 45th Street),
Hell's Kitchen
(212) 664-1507


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The Physician Assistant (PA) Section of the Society of Critical Medicine (SCCM) has identified the Surviving Sepsis Campaign (SSC) as a prime model for mentoring that improves patient care while affording PAs unique growth opportunities. This type of relationship can be applied to all professionals on the sepsis care team.
In this latest offering from the SSC webcast series, the PA Section will share examples of prime mentoring activities from the SSC. Presentations from the viewpoint of the mentee, mentor, and an objective educator will set the stage for participants to implement a mentoring program that can provide significant professional growth for all involved while furthering the reach of the SSC. Presenters include Ryan O'Gowan, MBA, PA-C, FCCM, from St. Vincent's Hospital, Mari Mullen, MD, from the University of Massachusetts, and Emanuel P. Rivers, BS, MD, MPH, IOM, from Henry Ford Hospital. During this webcast, faculty will:
- Describe the role of mentor and mentee in a healthcare setting
- Explain the benefits of serving as a mentor
- Outline goals for a mentoring relationship using the SSC as a model
The SSC as a Model for Mentoring webcast will be held on Tuesday, October 15, 2013, at 1:00 p.m. Central Time (view additional time zones).
Register online today. If you have any questions, please contact SCCM Customer Service at +1 847 827-6888.
Previous SSC webcasts and related educational slide presentations are also available on a variety of topics.
This webcast series is supported by the Gordon and Betty Moore Foundation
Surviving Sepsis Campaign Implementation Kits
The Surviving Sepsis Campaign has developed pre-packaged kits to help clinicians improve sepsis identification, management and treatment within their institutions. Each kit contains two posters featuring the graded recommendations from the latest guidelines, 25 pocket guides, 25 bundle cards and a package of 15 Surviving Sepsis Campaign logo pins. Visit www.survivingsepsis.org/improvement for more resources.


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Big Hunter 2.2.0 Mod Apk
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You can hunt animals successfully by attacking their heads.
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Download Big Hunter 2.2.0 Mod Apk
Note: All products on UnikTheme.com tested, without any malware or virus. You can always find the latest version here. If you liked this product, for support the developer Please buy it.


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108: Coffee with Katie Hays
Katie Hays and I had church. Church happened. And it's happening now, as you read these words. And it will happen again when you hit 'play.' Katie told us about church moments beyond the church hour and walls. She told us about … Continue reading 108: Coffee with Katie Hays


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|Publication number
|Publication type
|Application number
|Publication date
|Mar 9, 1999
|Filing date
|Sep 17, 1997
|Priority date
|Sep 17, 1997
|Fee status
|Publication number
|John R. Thompson
|Original Assignee
|Merck & Co., Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (1), Non-Patent Citations (13), Referenced by (24), Classifications (5), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention relates to a procedure for DNA-mediated transformation of Saccharomyces cerevisiae by electroporation. More particularly, the invention relates to the treatment of the host strain with lithium acetate and dithiothreitol (DTT) in order to weaken cell wall structure and increase the efficiency of transformation.
Electroporation has become the preferred method for gene transfer due to its ease and efficiency of operation in comparison to alternate techniques. To date, electroporation has been utilized to transform a wide variety of cell types including mammalian cells, plant protoplasts, bacteria, fungi and yeast. The technique involves subjecting cells to a high voltage electric field, which results in the temporary formation of pores in the membrane, thereby allowing exogenous DNA to enter the cells. For example, laboratory strains of the yeast Saccharomyces cerevisiae have been transformed through electroporation with either self-replicating plasmids or by integration of linearized plasmid DNA in the host genome.
Yeast provide an attractive alternative to prokaryotes as host organisms. They can be used for large scale fermentations and are adaptable to continuous fermentation processing. In addition, they are not susceptible to phage infection and generally require only semi-sterile conditions. Conveniently, these cells may be immobilized for the production of metabolites and enzymes.
A number of yeast strains have been used as host organisms for recombinant gene expression.
References describing transformation of S. cerevisiae by electroporation include Hashimoto et al., Appl Microbiol Biotechnol (1985) 21:336-339; Karube et al., FEBS (1985) 182:90-94; Rech et al., Nucleic Acids Research (1989) 18:1313; Simon and McEntee, Biochemical and Biophysical Research Commmunications (1989) 164:1157-1164; Dolorme, E., Appl. Environ. Microbiol (1989) 55: 2242-2246; and Becker and Guarente, Methods in Enzymology (1991) 194: 182-187. Transformation of S. cerevisiae with alkali cations or thiol compounds is described by Ito et al., J. Biol. Chem. (1984) 48: 163-168; Brzobohaty and Kovac, J. of General Microbiology (1986) 132:3089-3093.
The present invention relates to a process for the transformation of Saccharomyces cerevisiae for use as a preclinical research tool. The invention also relates to the use of the transformed host to develop screens to detect new drugs.
The present invention relates to a novel process for the transformation of Saccharomyces cerevisiae by electroporation. More particularly, the invention relates to the treatment of the host strain with lithium acetate and DTT in order to weaken cell wall structure and effect an optimized yield of transformants.
The first aspect of the invention relates to the process which comprises (1) pre-treating the host strain with lithium acetate/DTT; (2) adding plasmid DNA containing a selectable transformation marker to the treated host strain; (3) introducing said DNA construct into the host strain by application of an electrical pulse to form transformed cells; and (4) selecting said transformed cells, in this case, by their ability to grow in medium selecting for the plasmid encoded marker.
A second aspect of the invention relates to the use of the transformed host strains as a preclinical tool.
A third aspect of the invention relates to the introduction of other genes into Saccharomyces that would be antifungal targets. For example, it is contemplated that introduction of genes from pathogenic organisms into S. Cerevisiae to develop Saccharomyces-based screens for inhibitors of gene products derived from pathogenic fungi is within the scope of the invention.
The transformation methodology described here represents an improvement over an established electroporation protocol in order to achieve a higher efficiency of transformation. Achieving the highest possible efficiency of transformation is of particular advantage in circumstances where large numbers of transformants are required or transforming DNA is in limited supply. For example, a mutant host strain is often transformed with a plasmid-based genomic library as a means to isolate and identify the wild-type gene corresponding to the mutant allele. Typically, tens of thousands of transformants are required in such an experiment and the library DNA is often in limited supply. Such DNA libraries can, in principal, be amplified to produce more DNA, however, this is undesirable because each round of amplification results in a degradation of the representation of the library and lessens the chance that the gene of interest will be found. Thus, any improvement in transformation efficiency reduces consumption of precious and often irreplaceable DNA preparations. Further, while many wild-type laboratory strains transform well with established methods, very often introduction of a desired mutation into a strain results in a concomitant reduction of the strain's transformation efficiency. In some cases, mutant strains can be completely refractory to transformation by established methods. The transformation procedure described here is particularly advantageous in these situations.
Various procedures for transforming Saccharomyces cerevisiae are currently in use. The original spheroplast method remains one of the most efficient methods in terms of yield of transformants per microgram of DNA. However, preparation of spheroplast competent cells is time consuming, relies on carefully controlled enzymatic digestion of the cell wall by crude preparations of glucanase and requires plating of transformants embedded in soft agar. Moreover, transformation by the spheroplast method often results in cell fusion events and the resulting ploidy variation can complicate downstream genetic analyses. These disadvantages of the spheroplast method provided the impetus for workers in the field to develop alternative methods.
A procedure based on chemical treatment with alkali metals such as lithium acetate was developed by Ito et al (J. Biol. Chem. (1984) 48: 163-168). This method requires fewer manipulations than the spheroplast protocol and allows direct spread plating of transformants on the surface of agar plates. Chen et al. improved on the lithium treatment protocol by the addition of DTT, a chemical known to release mannoproteins from the yeast cell wall (Chen et al., Curr Genet (1992) 21:83-84). Variations of the lithium treatment method are still in widespread use because of their relative simplicity compared to the spheroplast method. However, the best optimized lithium treatment protocols are less efficient than spheroplasting method.
Several electroporative methods for transformation of Saccharomyces have been developed. The first electroporation protocols, while even less laborious than the lithium treatment protocols, provided relatively low yields of transformants (Hashimoto et al., Appl Microbiol Biotechnol (1985) 21:336-339; Karube et al., FEBS (1985) 182:90-94; Rech et al., Nucleic Acids Research (1989) 18:1313; Simon and McEntee, Biochemical and Biophysical Research Commmunications (1989) 164:1157-1164; Dolorme, E., Appl. Environ. Microbiol (1989) 55: 2242-2246). However, Becker and Guarente published an optimized protocol for electroporation that resulted in a transformation efficiency greater than the original spheroplast methodology while preserving the simplified preparation procedures common to the electroporative methods. The electroporation protocol of Becker and Gaurente involves extensive washing of cells to reduce ionic strength and electroporation of cells as a concentrated cell suspension in an osmotically supported low ionic strength solution. Our protocol introduces a short pretreatment with lithium acetate and DTT prior to this washing procedure. This simple modification results in a 5-10 fold increase in transformation efficiency.
The transformed S. cerevisiae can be used to study mode of action of antifungals as well as to evaluate potential resistant mechanisms. They can also be used for developing screens to detect new drug candidates. Additionally, they can be used for molecular manipulation of S. cerevisiae for gene cloning and gene disruption experiments.
The protocol is generalizable to work with any yeast auxotrophic or antibiotic resistance marker (e.g. -his, -leu, -ura, -ade, -trp or G418 resistance).
The following examples are provided to illustrate the invention but are not to be construed as limiting the scope of the invention.
Strains and DNA were isolated and handled by standard procedures J. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning, A Laboratory Manual", second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)!, referred to as Maniatis. Media recipes and many of the procedures for working with S. cerevisiae are described in M. D. Rose, F. Winston, and P. Hieter, "Methods in Yeast Genetics: a Laboratory Course Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), referred to as MYG.
Two host strains of Saccharomyces cerevisiae were selected to demonstrate the improved efficiency of the new electroporation protocol. Strain YPH98 is a wild-type strain that generally transforms with high efficiency and possesses several common auxotrophic markers (MATα ura3-52 lys2-801, ade2-101 trp1-Δ1 leu2-Δ1). A second strain, R812-18 is a spontaneous mutant of YPH98 selected for resistance to an antifungal agent. R812-18 also shows reduced transformation efficiency with respect to its wild-type parent, YPH98. The plasmid used in this example is pRS316 and is a centromeric autonomously replicating plasmid carrying a selectable URA3 marker (Sikorski and Hieter, Genetics (1989) 122:19-27).
100 ml of YPAD medium were inoculated with a colony or 0.1 ml of a saturated starter culture. The culture was grown overnight at 28�-30� C. and cells were harvested when an OD600 of 1.0-1.5 was obtained. Cells were harvested by centrifugation at 3000 RPM and suspended in 25 ml of a LiAc/DTT/TE solution consisting of 100 mM lithium acetate, 10 mM DTT, 10 mM Tris pH 7.5, and 1 mM EDTA (filter sterilized). Cells were incubated in the LiAc/DTT/TE solution for 1 hour on ice with occasional gentle mixing. The cells were harvested at 3000 RPM and washed twice with 50 ml of ice cold sterile water and once with 10 ml ice cold 1M sorbitol. The final cell pellet was suspended in a volume of 1M sorbitol equal to the wet weight of the cell pellet thus forming a 50% suspension of cells (typically ˜0.2-0.4 ml).
It is believed that the DTT can be used in a range of about 10 mM to about 100 mM.
10-100 ng of DNA in 1-5 μl of water or TE buffer was placed in a sterile screw cap microfuge tube. Electrocompetent cells (50 μl) were added and mixed. The mixture was transferred to a pre-chilled 0.2 mm Biorad electroporation cuvette. Electroporation was performed with a BioRad Gene Pulser instrument set at 1500v, 25 μF, and 200 Ω. The resulting time constants reported by the instrument were 4.5-5.0 ms. The electroporated samples were immediately diluted with 1 ml of ice cold sterile 1M Sorbitol and returned to ice. Aliquots of the transformation mix were either plated directly or diluted as needed with additional 1M sorbitol and spread onto the appropriate selective medium. The transformants were incubated 3-5 days at 28�-30� C. to allow colony formation.
Electrocompetent YPH98 and R812-18 cells were prepared as outlined above. To illustrate the benefit of the lithium acetate/DTT incubation, two batches of cells were prepared from each strain and the lithium acetate/DTT pretreatment was omitted for one of the batches. 50 μl aliquots of each electrocompetent cell preparation were electroporated as described above after mixing with 100 ng of pRS316 DNA in 5 μl of TE buffer (10 mM Tris, 1 mM EDTA pH 7.5). The electroporated cells were serial diluted, plated on synthetic complete medium lacking uracil and incubated 4 days at 30� C. to allow colony formation. The resulting transformants were counted on dilution plates that yield between 90 and 400 colonies per plate. The results, expressed as transformants per microgram of DNA, are tabulated below.
______________________________________ Transformation RelativeStrain Pretreatment Frequency Increase______________________________________YPH98 none 1.25 � 10.sup.5 1.0YPH98 Lithium 1.41 � 10.sup.6 11.3 Acetate/DTTR812-18 none 1.62 � 10.sup.4 1.0R812-18 Lithium 9.25 � 10.sup.4 5.7 Acetate/DTT______________________________________
The relative increase listed reflects the increase in transformation efficiency directly attributable to the lithium acetate/DTT pretreatment. While the absolute transformation efficiency of the mutant strain R812-18 is about 8-fold lower than its wild-type parent, both strains showed a similar relative increase in efficiency as a result of the lithium acetate/DTT pretreatment.
This example serves to illustrate the contribution of both lithium acetate and DTT to the overall increase in transformation efficiency. Experimental procedures were identical to Example 1 with the exception that pretreatment during competent cell preparation was varied to consist of no pretreatment, pretreatment with lithium acetate only (100 mM lithium acetate, 10 mM Tris, 1 mM EDTA pH 7.5), pretreatment with DTT only (10 mM DTT, 1 mM EDTA pH 7.5), and standard pretreatment with lithium acetate/DTT (0.1M lithium acetate, 10 mM DTT, 1 mM EDTA pH 7.5). A culture of YPH98 was harvested separated into 4 aliquots and electrocompetent cells prepared with each of the 4 described pretreatments. The plasmid used for transformation in this experiment is YEPlac195 (Gietz and Sugino, Gene (1988) 74:527-534). The plasmid is a multi-copy yeast plasmid bearing a 2 μM origin of replication and the URA3 gene as a selectable marker.
Cells (40 μl) from each of the four different pretreatments were electroporated with 100 ng of YEPlac195 using the same electroporation conditions as Example 1. The electroporated samples were serially diluted, 0.1 ml aliquots spread plated on synthetic complete medium lacking uracil and incubated 4 days at 30� C. Colony counts were collected from dilution plates with between 100-250 colonies and used to calculate the transformation efficiencies. The results are tabulated below.
Because lithium acetate and DTT treatments have been shown to stimulate DNA uptake in the absence of an electrical pulse, we tested the dependence of our transformation protocol on the electrical pulse. A sample of lithium acetate/DTT pretreated electrocompetent cells was mixed with DNA but not subjected to an electrical pulse before plating on selective medium. This sample yielded zero transformants (<1 transformant/10 ng DNA) and illustrates the importance of the electrical pulse to the success of the procedure. The lack of even a low frequency of transformants from the lithium acetate/DTT treated cells in the absence of a pulse is not unexpected however. The non-electroporative lithium acetate methodology for transforming yeast relies on a treatment of the competent cell/DNA mixture with concentrated polyethylene glycol solution to induce DNA uptake and generally calls for the addition of a 10 to 100 fold greater amount of DNA to the reaction. No polyethylene glycol is involved in our procedure.
______________________________________ Transformation RelativePretreatment Frequency Increase______________________________________None 2.38 � 10.sup.4 1XLithium Acetate 9.30 � 10.sup.4 3.9XDTT 9.3 � 10.sup.4 3.9XLithium Acetate + DTT 2.8 � 10.sup.5 11.8X______________________________________
Examination of the results shows that pretreatment with either lithium acetate or DTT alone showed some increase in efficiency. However, the combination of both lithium acetate and DTT in the pretreatment results in the highest transformation efficiency conferring roughly an order of magnitude increase over no pretreatment.
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|Citing Patent
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|Feb 22, 1999
|Mar 21, 2000
|Method for more efficient electroporation
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|Genetronics, Inc.
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|Genetronics, Inc.
|Apparatus for electroporation mediated delivery of drugs and genes
|Jul 13, 1999
|Oct 16, 2001
|Genetronics, Inc.
|Method and apparatus for electrically assisted topical delivery of agents for cosmetic applications
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|Jan 15, 2002
|Method for more efficient electroporation
|May 18, 2001
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|Method and apparatus for electrically assisted topical delivery of agents for cosmetic applications
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|Aug 4, 2009
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|Apr 12, 2011
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|Greener Alan L.
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|Jul 29, 2004
|Edward Nolan
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|Apr 28, 2004
|Oct 14, 2004
|Hofmann Gunter A.
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|David Rickwood
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|U.S. Classification
|435/6.13, 435/483
|International Classification
|Cooperative Classification
|European Classification
|Nov 18, 1998
Owner name: MERCK & CO., INC., NEW JERSEY
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:THOMPSON, JOHN R.;REEL/FRAME:009607/0407
Effective date: 19970124
|Sep 25, 2002
|Maintenance fee reminder mailed
|Mar 10, 2003
|Lapse for failure to pay maintenance fees
|May 6, 2003
|Expired due to failure to pay maintenance fee
Effective date: 20030309


text:
Summer Theater Workshop
This summer we will be creating our vaudeville acts with pirates in mind...ay' mateys - yes that's right, pirates!! We will learn some pirate songs, try our hand at pirate themed skits, and perhaps some stage combat skills. Our pirates will also probably unicycle, tumble, dance and juggle too in this two-week workshop.
For children ages 8-13 (CITs age 14-18).
Weekdays July 10 - 21 , 2017
July 10 - 21, 2017
Mon-Fri. 9:30-3:30 daily
Final performance for family and friends at 2:30 pm on Friday, July 21st.
Grace Vision Church (formerly St. John's), 80 Mt. Auburn Street, Watertown, MA


text:
Massa tops practice for Chinese GP
Felipe Massa put Ferrari at the top of the timesheets yesterday, setting the fastest lap in practice for Sunday's Chinese Grand Prix.
The Brazilian set a time of one minute 35.340 on the soft tyre, over a tenth of a second quicker than Kimi Raikkonen's Lotus, with Fernando Alonso in the second Ferrari over four-tenths of a second slower than his team-mate in third.
"I was not really happy in the beginning on the medium tyres when we were not even so quick compared to other cars. But looking how much we improved with the soft tyres, I think that was maybe amazing," Massa told reporters in the Shanghai paddock.
He continued: "I think I did a good lap on the softs. I think that's positive and the degradation on the tyres for the race, I think the long run was not bad. "So, confident for the weekend. I hope we can do everything we did today, tomorrow and on Sunday as well."
Massa's session-topping time deposed Mercedes' Nico Rosberg from the top of the timesheets. Both Mercedes looked quick yesterday, with Rosberg heading teammate Lewis Hamilton for a Silver Arrows one-two in the first session.
Rosberg, who won here last year, initially seemed to have carried that speed over into the second session but the German eventually ended the day fourth fastest ahead of Mark Webber who was the best of the Red Bulls in fifth.
Jenson Button ended the day sixth in the McLaren ahead of former teammate Hamilton. The Force India duo of Adrian Sutil and Paul di Resta were next, while Sebastian Vettel, winner of the last race in Malaysia, rounded out the top ten.
Not for the first time this year, talk after practice was all about the tyres. Pirelli have brought their medium and soft compound tyres to China, with the latter being used for the first time this year and falling apart after only a few laps, prompting complaints from drivers. But the softer tyre is much quicker over one lap compared to the medium, which is the preferred race tyre.
This is likely to put teams, especially those that make it into the top ten but aren't realistically fighting for pole, in a dilemma. Do they choose not to run in the final part of qualifying so they can then opt for the medium tyre to start the race on, or do they opt for the soft and try and start as high up the grid as possible even if it means they would have to stop early on in the race?
"Maybe, yes, some will try," Force India's Adrian Sutil said after practice. "I expect no one going longer than six to eight laps (on the soft tyre)," the German said.


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Most Helpful Guy
Yes. ALL guys do it. I consider myself nice and respectful and, as hard as I try not to look, I end up looking. I don't try to leer and I certainly do not see women as objects but I'll be damned if I won't look at a nice body. We *are* animals after all and it is our primal instinct to look.82|60|0
Wow intresting way of saying it.


text:
A dog followed his nose to three puppies that were tossed out Monday with garbage at a Bay Area park.
A couple were walking their Australian shepherd at Paso Nogal Park in Pleasant Hill when its nose alerted them to a tightly wound plastic bag, Contra Costa animal control officials said.
PHOTOS: Memorable animal stories
They untied the bag and found three small puppies who officials said "had been discarded like trash."
The couple took the puppies home with them cleaned them up, and fed them. The following day, they dropped the puppies off at the animal shelter.
"We are both saddened at the inhumanity and grateful at the compassion shown," officials said.
The puppies were transferred to the East Bay SPCA in Dublin.
Anyone with information about the puppies' owner is urged to call animal control at (925) 335-8300.
For breaking news in California, follow @VeronicaRochaLA


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Download Wedding Gown Rental apk 4.0.2 free for Android smartphone
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