My desired gene size is nearly1000 bp. when I use pfu DNA polymerase and taq mastermix the PCR product size is 1000bp but when I use taq DNA polymerase (both Roche and Qiagen) it is 1500 bp.is there any reason for this result?
In brief, no there isn't any reasonable reason. A number of the things you describe should not be happening. Random false amplification should be shorter, not longer, because long sequences amplify less efficiently than short sequences. And the difference between Taq pol vs. Taq mastermix is just bizarre. I guess it could come down to a minor difference in reaction buffer composition, but this is definitely not normal.
I'm almost tempted to guess that a change in template is more likely. Were these done on different days with different starting materials? Also, perhaps some change, like a recombination or secondary structure happened to DNA that was stored for some time.
If you can actually reproduce this from fresh materials, rest assured that this is a very weird result, and there's nothing wrong with your understanding. In that case you'll just have to pick the conditions that work, and quit working with that particular Taq pol preparation.
In general shorter sequences amplify more readily than long ones, but if the longer amplicon has a nucleotide composition and/or sequence (fewer blocks or runs of a particular base and/or less potential for alternate 2ndary/tertiary structure) even just slightly more easily traversed/replicated by a given polymerase /rxn mix combination, it can easily become the dominant product over the shorter intended target.
This is why there is a market for such a wide array of PCR polymerases and rxn mixes --primers are only the beginning of a high-yield/high-specificity PCR .
Try sequencing your different products and/or cross-doping 2nd-round PCRs (ie dope and / or template a pfu/taq-mastermix rxn with 1500bp product from your Roche/Qiagen Taq rxns and try Taq rxns using 1000bp products from pfu/taq-mastermix PCRs). The former procedure will rigorously identify the correct product, if any, and whether the larger amplicon is an intrinsic or contaminating natural genomic sequence vs one artifactually generated during early rounds of the PCR. The latter experiment will shed light on the role of relative template efficiency vs polymerase type in generating your initial anomalous results.
It would help if you told us what the reaction conditions of your PCR reaction were (i.e. Tm of primers, annealing temperature, and extension times). Taq is a fast enzyme, and is well capable of amplifying ~1500 bp in a little over 30 seconds. I'm wondering if there is a secondary binding site for your primers which produces a product during PCR that then gets degraded by the exonuclease activity of pfu. Do you see any low molecular weight smearing along with the 1000 bp product when you use the Taq/Pfu master mix?
I use same program for all of reaction.
Initial denaturation 94°c : 5min
Cycles(30)
Denaturation 94°c : 60sec
Annealing 61°c : 60sec
Extension/elongation 72°c : 90sec
Final extension/elongation 72°c : 7min
Hold 4°
And in this picture you can see result of pfu in left(1000bp) and taq in right(1500 bp) of picture.In PfU reaction always I get stong bond but in taq polymerase I get weaker bond.
Hi there,
Is it really important to get the reason? I guess what you are interested in is to have the sufficient amount of the expected DNA for downstream experiments. Obviously the 1kb product is the one you were looking for and as it has been synthezised with Pfu polymerase the risk of mutation is much lower compared to Taq amplified product. Just make sure at the end that the sequence fits exactly with the expected one.
- Badges
- Science method