Dye in PCR Buffer - Inhibiting downstream techniques? - ligation/digestion/cloning (Oct/04/2010 )

Hello,

I have used GoTaq Flexi Green in my PCR reactions. I was wondering if anyone knows if this should inhibit downstream processes such as ligation and digestion. I have made my PCR product long enough to digest directly, then put into pCDNA. I get a few background colonies, but no colonies from the PCR insert. It would seem that maybe the buffer is inhibiting ligation? To much glycerol perhaps for a digestion, then a ligation? However the ligations works fine if you just TA clone directly into PGEM. I am suspicious. Anyone have any thoughts?

I used to use Promega GoTaq Flexi Green. Did TA cloning with it as well (after gel purification, of course). No issues.
Hope you didn't directly use the green pcr solution and do your RE...

Adrian

adrian kohsf on Tue Oct 5 02:48:00 2010 said:

Why couldn't one use the PCR product directly with the RE? I think the issue is to much glycerol. A high enough dilution of the PCR product be ok.

Yup, probably due to glycerol. Besides, I do think that the green and yellow dye "might" have something to do with affecting the RE activity. Also, you "taq" is still there... wouldn't it be another competitive inhibitor of the RE activity? Once your RE cut it, the taq sort of like anneal it back, possible? I can't answer that but just speculate.

If your enzyme cuts and leaves a 5' overhang, then the PCR enzymes will extend, destroying your ability to clone the cut fragment into a vector. Purify your PCR product before you cut with the restriction enzyme.

I use GoTaq mastermix from promega with green dye as well. After PCR the fragment, I purify that using Qiagen PCR purify before cut them with RE. After digestion, I do another purification from enzymatic reaction. At this stage, it is ready to do ligation. Once ligation done, I don't purify it, just go straight to trasformation.

phage434 on Tue Oct 5 18:57:12 2010 said:

I don't know. TAQ would have to have room 37 degree activity. Does it?

Yes. Remember that adding a single base will destroy your site. Most reactions slow down 2x for each 10 degrees lower temperature. So, we're talking about an 8x - 10x reduction in speed. The speed at 72C is > 1Kb/min, so adding a single base will not take long.