Reamplification after agarose gel extraction
Posted 18 April 2009 - 03:29 AM
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!
Posted 18 April 2009 - 06:10 AM
Posted 19 April 2009 - 04:49 AM
Try diluting your extract 1:1000 and use that as template.
Posted 29 April 2009 - 07:33 AM
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.