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Reamplification after agarose gel extraction


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#1 OA17

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Posted 18 April 2009 - 03:29 AM

Hello!

I have been having quite a few problems for reamplifying a purified band after running the PCR on an agarose gel. I don't know if the problem is due to a bad purification or due to a bad primer annealing because of template degradation at the edges.

I have tried to purify the band using different methods such as QuiaexII, MinElute gel extraction kit, freeze and squeze, but have had no good results: I'm not able to reamplify at all.

I can only reamplify the DNA if I clean the PCR without passing through gel (for example with MinElute PCR purification kit).

Has someone had this problem? Does someone have a solution?

Thanks in advance!

#2 phage434

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Posted 18 April 2009 - 06:10 AM

The main thing to remember about PCR is that it needs extremely small amounts of template. In principle, a single molecule is sufficient. The usual problem people have is that they add too much template, usually in too high a volume of liquid. This introduces inhibitors to the PCR reaction, and it fails. My suggestion is that you avoid all of the gel purification issues by taking a pipette tip, plunge it into the gel at the correct location, and then into your PCR tube. Make sure you do 35+ cycles.

#3 molgen

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Posted 19 April 2009 - 04:49 AM

I agree with phage. Too much template.
Try diluting your extract 1:1000 and use that as template.

#4 OA17

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Posted 21 April 2009 - 08:41 AM

Thanks a lot, I will follow your advice!

#5 hanming86

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Posted 29 April 2009 - 07:33 AM

Another thing is instead of reamplifying it urself

Do a TOPO TA cloning . let E.coli amplify it in-vivo for you.

the gel extracted fragment should be fit for a TA cloning assuming that there's still some template with non-degraded end plus A tail.
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