I used Hi-Fi Taq (invtrogen) to amplify a 1kb fragment from HIV-1. The PCR product was so viscous that I couldn't suck it up a pipette tip, and the subsequent agarose gel is negative. The solution was clear and not whitish and stringy as you would expect if the DNA strands were disintegrated. I have observed this only when amplifying cDNA as the same reactions and conditions work perfectly for DNA. I use the RT kit from Applied Biosystems which comes with RNAse inhibitor for cDNA synthesis. The cDNA is stored at -20deg until use. Anyone with any idea what might cause this?
I am assuming the reaction was not viscous at the beginning of the PCR reaction, try a control reaction with no added Taq, and a second control with no added forward primer, and see what happens.
In my experience the reaction mix is "not" viscous to start with, or after 40-50 cycles, when using a heated lid to prevent evaporation.
Usually the reaction buffer is viscous that;s why you have vortex very well before use,
double check your buffer, buffer of cDNA synthesis and buffer of Taq enz amplification.
Have you considered excessive evaporation during thermocycling? This occurs quite frequently.
I know what you are talking about, but unfortunately I have no answer!
I have been amplifying a 9kb HIV-1 fragment from cDNA until recently whenmost of the wells end up with some colorless viscous product that is so thick it won't go through a pipette tip! This does not happen when I amplify 2kb GAG from the same cDNA. Did you ever figure out how not to get it?
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