4 replies to this topic

[OA17]

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!

[phage434]

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.

[molgen]

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

[OA17]

Thanks a lot, I will follow your advice!

[hanming86]

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.