DNA Polymerase I, Large (Klenow) Fragment

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.

Yes. There is a phosphate group on the 3' end of all TaqMan probes that prevents such extension.

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).

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.

The fidelity of this PCR enzyme is affected in two ways. First, AmpliTaq DNA Polymerase typically binds to and incorporates base analogs less efficiently than conventional dNTPs, which means that polymerase activity is lower in reactions that contain base analogs. Second, the analog may pair with more than one conventional complementary template base, so the analog may be incorporated at an increased level compared to conventional dNTPs. For the best fidelity, we recommend that base analogs are included at low concentrations in the reaction.