What enzymes can I use to convert a molecule with a 5' or 3' overhang to a blunt molecule? What is the main difference between them?
T4 DNA polymerase and Klenow fragment of E.coli DNA polymerase can both convert overhangs to blunt molecules. The 3' - 5' exonuclease activity of the T4 DNA polymerase is much more efficient than Klenow.
Is there anything to prevent AmpliTaq Gold DNA polymerase from extending from the 3’ end of a TaqMan probe in a 5’ nuclease assay?
Yes. There is a phosphate group on the 3' end of all TaqMan probes that prevents such extension.
How does AmpliTaq Gold DNA Polymerase differ from AmpliTaq DNA Polymerase?
AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase that contains a proprietary chemical (or so-called hot start molecule) bound to the enzyme's active site. In order to activate the AmpliTaq Gold DNA Polymerase fully, we recommend an initial activation step of 95 degrees C for 10 min when using GeneAmp 10X PCR Buffer I and/or GeneAmp 10X PCR Buffer II and Mg in one of our thermal cyclers. When using GeneAmp 10X PCR Gold Buffer, activation time can be reduced to 5 minutes. Once activation is complete, you can proceed with your standard PCR cycling program (denaturing, annealing, extension, etc).
Does AmpliTaq Gold DNA Polymerase contain exonuclease (proofreading) activity?
No, AmpliTaq Gold DNA polymerase does not contain proofreading activity, however fidelity in PCR amplifications utilizing this enzyme may be improved. High fidelity can be achieved by: 1. Decreasing the final concentration of each nucleotide to 40-50 uM. 2. Using the lowest MgCl2 concentration possible. 3. Using less enzyme. 4. Decreasing extension times. 5. Using the highest annealing temperature possible. 6. Using as few cycles as possible.