polymerases for PCR - TOPOTAQ (Aug/29/2006 )
I hope somebody can give me an advice - I am gonna do PCR of my really small quantity of cDNA. The protocol is optimized for TOPOTAQ, but I was wondering if I can't use another polymerase, like Ampli Taq Gold, Ex Taq, KOD+ or ...
what is it "reallysmall" quantity of cDNA ( strongly expressed gene or non-expressed atall ?
the word "quantity" usually has a kind of "number" cDNA shurelly is not in "microliters "
cDNA - usually consided " a simple template" for PCR like "plasmidPCR" - small ammounts
may need "carrier" nucleic acids in pcr (tRNA etc)
Make sure to use taq for it is far more robust than most proof-reading enzymes (proof-reading enzymes do not like dUTP in the mix, which frequently is left over after reverse transcription).
I would take more care in how you purify your DNA than the $$$ polymerases.
Choice of polymerase also depends on what you want to do after amplification. Do you intend to clone after amplification?
Well, I use 5 ng of total RNA as starting material for cDNA synthesis, so it is really small amount. Im planning to insert it in vector afterwards.
What can you tell me about differences among polymerases TOPOTAQ, KOD+, Ex Taq and AmpliTaq Gold???
Hmm... I have never played with TOPOTAQ, or AmpliTaq Gold before.
More info would be nice. Like how big is the cDNA that you would like to amplify?
However, here is a pointer in the right direction. look up a paper called
Amplification Efficiency of thermostable DNA polymerases" by Bahram Arezi, Weimei Xing, Joseph A. Sorge and Holly H. Hogrefe.
If this cDNA is very important perhaps you might want to invest in some dUTPase (www.genwaybio.com) dUTPase increase the efficiency of the PCR reaction. High fidelity polymerase stall when they encounter dUTP, stalling leads to increased probabilty of an abortive reaction.