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Re: how to add a "A" at PCR product ends

There is now a listing on the OpenWetWare Wiki about adding 3' A overhangs using Taq polymerase. The site listing can be found here: http://openwetware.org/wiki/Addition_of_3%27_A_overhangs_to_PCR_products In order to overcome the fact that Taq will add ...

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by CarmeanC
Fri Mar 09, 2012 6:38 pm
Forum: Molecular Biology
Topic: how to add a "A" at PCR product ends
Replies: 6
Views: 22416

Colony PCR

... this ~4kb gene using Phusion polymerase. I was able to amplify the gene. I then purified it using the Qiagen Purification Kit and added 'A' overhangs to the product since Phusion produces blunt end products. Once I added the 'A' overhangs (cycling conditions of 72 degrees for 25 minutes), ...

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by claudianisha
Fri Dec 02, 2011 1:59 am
Forum: Molecular Biology
Topic: Colony PCR
Replies: 4
Views: 4822

Cloning bacteriphage lambda fragments

... be able to insert into the plasmid, except those at the left and right ends of the bacteriphage genome. For example the fragment with HindIII overhangs on either side would ligate to a fragment with HindIII-EcoRi overhangs, and then that would ligate into the plasmid. But according to the ...

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by francisp
Tue Oct 25, 2011 12:26 am
Forum: Molecular Biology
Topic: Cloning bacteriphage lambda fragments
Replies: 0
Views: 1206

PCR DNA in one direction?

... sense, the replies I understand. Hmmmm. Primer design is one answer but you are missing the obvious: RE analysis. Whether you incorporate cloning overhangs in your primer or not, approx 50% of the clones will be in the direction you wish. So just pick a few of the ones which show the predicted ...

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by merv
Sun Oct 02, 2011 7:55 pm
Forum: Bioinformatics
Topic: PCR DNA in one direction?
Replies: 6
Views: 6792

Features of the leading strand synthesis on DNA ends

... colleagues! Help me please with one question. Have the DNA polymerase can complete leading-strand synthesis in the ends of DNA, or no, leaving 5'-overhangs? I am interesting in the problem of DNA end replication, and in some literature I have find a data, describing two mechanisms for this problem ...

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by carterjer3
Wed Sep 28, 2011 11:56 am
Forum: Molecular Biology
Topic: Features of the leading strand synthesis on DNA ends
Replies: 2
Views: 3944
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