Effects of Polymerases on DNA Footprints

[JB04]

Hello,

for my Masters thesis I'm doing a Footprinting experiment with HeLa cells. For this the DNA is cut at unprotected sites where no proteins are bound. Via 2 PCR steps the fragments are amplified and then analyzed by capillary gel electrophoresis.

 

Most protocols use Vent polymerase for the whole procedure because it creates blunt ends which is important in the first PCR step of the procedure. However I wasn't able to get good results with Vent, so instead we used iProof to create blunt ended products, and iTaq for the 2nd amplification step because of its higher effectivity.

 

My question now would be if iTaq adds an A at the end of the fragments like the ordinary Taq does, and if yes in how many % of fragments this is the case? Also does a single bp overhang even affect the separation of the fragments?

 

It would be great if someone has experience with this topic, and could help me out

 

Kind regards

 

P.S.: I'm new here, I've used the search function but couldn't find matching results.

[hypervalent_iodine]

I can't comment about whether or not iTaq adds A's, but a 1 bp difference seems like it wouldn't be noticeable on a gel.

[CharonY]

I would just take a look at the spec sheet, but I doubt that they have somehow added proof reading to the polymerase. And as hyper said, one base is way way below the resolution limit of agarose (but not acrylamide) gels.