I amplified human gene cDNA(600 bp) with Q5 pol but after amplification I got 500 bp band with some nonspecific bands of less than 500 bp, then I performed gradient PCR and again I got 500 bp band with some non-specific bands.
When I used same cDNA template with Taq pol then I got only 600 bp band.
Q5 is more efficient than Taq pol then why it is not giving desired length and specific band of amplified cDNA ?
Q5 and Taq buffers are completely different, easy to see when you use NEB Tm calculator with your primers selected either Taq or Q5. You may see up to 10C difference in Tm whether you use Q5 or Taq. My guess is that you have the two variants in your cDNAs, oen amplify better in one buffer (due to Tm) and the other better in the other. As John sugegsted why bother with Q5 if taq works. If you are afraid of point mutation yuo can sequence before subcloning.
I do not know which gene it is but could you have more than one splicing form ? Easy to check on your gene model.
Q5 is intended for long amplicons and high G/C content. It doesn't mean that it's automatically better at everything. If it's possible to use Taq, then I would prefer Taq.
Ok Jhon but I have successfully amplified 200 bp gene with Q5 pol and this time I am facing problem with 600 bp gene why?
Q5 and Taq buffers are completely different, easy to see when you use NEB Tm calculator with your primers selected either Taq or Q5. You may see up to 10C difference in Tm whether you use Q5 or Taq. My guess is that you have the two variants in your cDNAs, oen amplify better in one buffer (due to Tm) and the other better in the other. As John sugegsted why bother with Q5 if taq works. If you are afraid of point mutation yuo can sequence before subcloning.
Hugo FTW.... Thanks! I never noticed this before...
untill today using same tm with neb 2x taq MM and Q5 has always worked for me...
sometimes i will optimize difficult pcr reactions in the cheap taq if im wasting abunch of Q5 on bad reactions, then go back to the Q5 for seq....
now i see in neb tm calculator how big of a difference optimal annealing temp is. ... 15'C difference for my primer pair between the 2 diff master mixes.
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