Long and Accurate PCR - (Mar/19/2007 )
Can anyone recommend a good (working) protocol for amplifying a large sequence of plant DNA? What are the fundamental parameters to consider for obtaining a long PCR product (from 10 to 40 kb)? I have already used Accutaq (SIGMA) but the supplier protocol worked on lambda phage DNA but nothing came from the tomato plant genome. Though I followed many of the tips they suggested, I had no resolution of the problem.
thanks a million for any suggestions!
I'd recommend the Roche Expand system. It worked for me with 20Kb+ sequences.
Low extension temperatures (66-68), short denaturing times (15-20 s).
aside for phage434 suggestions, (phusion is another example of long ranger polymerases)
DNA, quality is very important. Especially for plants, make sure it is clean (phenolic, polysaccarrides, etc). If possible try running a trial PCR, using primers you know work on the sample of DNA, to show that the DNA is actually clean enough to support PCR.
make sure your primers have very close annealing temperature (I am for tm of 58 Celsius). Use a primer calculator.
also increasing the extention time with every cycle helps.
adding more magnesium ions
1 - 5% glycerol helps too.