International Scholarships and Financial Aid

Wednesday, 13 June 2007

Overlap Extension PCR

Overlap Extension PCR is used to create long DNA fragments from short ones.

or used for Engineering the replication of target DNA through cloning, or changing its genetic code through mutations

PCR amplify the necessary fragments, using polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temp.
Clean up or gel extract the correct size band. Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template.
Use proofreading enzyme for extension. Run 3 reactions of 10,15 and 30 PCR cycles without end primers. (Template extension step) Add end primers, then continue cycling for another 15-20 rounds. Gel extract the correct fragment. Clone into a your desired vector.

check out the latest Nature Methods Protocol
http://www.nature.com/nmeth/journal/v4/n5/pdf/nmeth0507-455.pdf

13 comments:

Anonymous said...

Hi there. Sorry to hassle you but I ran across your site and
noticed you happen to be using the exact same template as me.

The only issue is on my site, I'm unable to get the theme looking like yours. Would you mind contacting me at: normaallan@aol.com so I can get this figured out. By the way I've bookmarked your
web-site: http://www.blogger.com/comment.g?blogID=984222275347260670&postID=6309778353600078468 and will certainly be visiting
frequently. Thank you!

Also visit my blog post; sophistry
Here is my web blog :: furze

Anonymous said...

This design is incredible! You certainly know how to keep
a reader amused. Between your wit and your videos, I was almost moved to start my own blog (well, almost.

..HaHa!) Wonderful job. I really loved what you had to say, and more than that, how you presented it.
Too cool!

Visit my web site; driveway austin
Here is my web-site : backup

Anonymous said...

Good day! I could have sworn I've been to this site before but after reading through some of the post I realized it's new to me.
Nonetheless, I'm definitely glad I found it and I'll be bookmarking and checking back often!


Here is my web-site ... pure

Anonymous said...

Hey there! My name is Michel and I just wanted to say your website rocks!
It's surprising due to the fact I use to have a blogging site that nearly had an identical url: http://www.blogger.com/comment.g?blogID=984222275347260670&postID=6309778353600078468 mine was only a few letters different. In any case, I am a big supporter of your website and if you ever want a guest post make sure you email me at: vernadelong@hotmail.com. I take pleasure in writing!

Also visit my web-site :: anaesthesia

Anonymous said...

Incredible! This blog looks just like my old one!

It's on a totally different subject but it has pretty much the same page layout and design. Wonderful choice of colors!

Feel free to visit my website :: tree removal

Anonymous said...

I like what you guys are usually up too. This kind of clever
work and reporting! Keep up the terrific works guys I've incorporated you guys to my blogroll.

Here is my web site ... removal chemical

Anonymous said...

Howdy. I am wondering if you'd be interested in doing a link exchange? I see your blog: http://www.blogger.com/comment.g?blogID=984222275347260670&postID=6309778353600078468 and my website are structured around the same subject matter. I'd love to switch links
or perhaps guest author a article for you. Here is my personal contact:
jrg-crawley@yahoo.de. You should contact me if you're even slightly interested. Appreciate it.

Feel free to visit my homepage :: pakerice
my web site :: automatske

Anonymous said...

Heya are using Wordpress for your site platform? I'm new to the blog world but I'm
trying to get started and set up my own.
Do you need any coding knowledge to make your own blog?
Any help would be really appreciated!

Also visit my web site ... grinding
my web site > taylors tree

Anonymous said...

I have gone ahead and added a hyperlink back to your internet site from one of my clients
requesting it. I've used your internet site URL: http://www.blogger.com/comment.g?blogID=984222275347260670&postID=6309778353600078468 and blog title: Blogger: Oxford Laboratory Technology to be certain you get the correct anchor text. If you woud like to check out where your hyperlink has been placed, please e mail me at: francisjeter@gmail.com. Thanks

My blog post :: web

Anonymous said...

Hey. I'm sorry to hassle you but I ran across your blog website and discovered you're using the exact same
template as me. The only issue is on my blog, I'm battling to get the layout looking like yours. Would you mind contacting me at: corey.waterman@fastmail.us so I can get this figured out. By the way I've bookmarked your
web-site: http://www.blogger.com/comment.g?
blogID=984222275347260670&postID=6309778353600078468 and will be visiting frequently.
With thanks!

My weblog :: link

Anonymous said...

You have made some decent points there. I checked on the net for more info about the issue and found most individuals will go along
with your views on this website.

My blog :: ideal waist to height ratio

Anonymous said...

My partner and I absolutely love your blog and find nearly all of your post's to be just what I'm
looking for. can you offer guest writers to write content to suit your needs?
I wouldn't mind writing a post or elaborating on a number of the subjects you write regarding here. Again, awesome weblog!

Take a look at my web page stump

Anonymous said...

Howdy terrific website! Does running a blog like this
take a massive amount work? I have virtually no understanding
of computer programming but I was hoping to start my own blog in the near future.

Anyway, if you have any recommendations or tips
for new blog owners please share. I understand this is off topic but I simply needed
to ask. Thanks a lot!

Here is my homepage: concrete concrete

Science Protocols