Can I use DMSO and Tween-20 in a PCR with an enzyme activation step of 94oC for 10 min? Or is it better to use an enzyme that doesn't need heat activation?
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#2
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Posted 17 March 2003 - 08:33 AM
Could you please write the purpose of your experiment? I have used formamide instead of DMSO to lower the annealing temperatures and it worked very well.
#3
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Posted 18 March 2003 - 09:45 AM
The purpose of my experiment?? It's a PCR, thus amplify a genomic sequence.
I am just wondering whether I should use AmpliTaq or AmpliTaq gold (the latter needs an 10 min 94 oC activation step). If the two reagents are not thermostable, they might degrade during this extended heating step??
I have been trying to amplify a region of APOE forever! Tried the methods in the literature step by step, still doesn't work (while I never have problems with my PCRs).
The latest methods requires 0.1 ml/L Tween, 100 ml/L DMSO and 20 mM ammonium sulfate.. still not working >
I am just wondering whether I should use AmpliTaq or AmpliTaq gold (the latter needs an 10 min 94 oC activation step). If the two reagents are not thermostable, they might degrade during this extended heating step??
I have been trying to amplify a region of APOE forever! Tried the methods in the literature step by step, still doesn't work (while I never have problems with my PCRs).
The latest methods requires 0.1 ml/L Tween, 100 ml/L DMSO and 20 mM ammonium sulfate.. still not working >
#4
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Posted 21 March 2003 - 02:41 PM
I have used 5% DMSO. It works.
