Afternoon everyone,
I am currently using Q5 polymerase from NEB to add restriction sites to an insert in a pBabepuro plasmid (~5400 Kb vector and 2.2 Kb insert). I have designed the primers to target the first 20 BP and last 20 BP of the insert, the restriction sites are added before and after the insert along with a 6 BP cap on each. The primers have been checked for hairpin formation and dimerization. I am using a standard set up, 2mM Mg++, 1uM primer, 200 uM dNTP, 10-50ng plasmid, and 0.5 uL Q5 polymerase in 50 uL.
For the PCR reaction 98C initial denaturing for 30s, then 30 cycles - 98C 10s, 62C 20s, 72C 1min, then final extension at 72C for 5 min.
I seen a faint band at 700 and 1200bp when I run the product out on a gel, I expect to see a 2.2kb product. There is faint smearing at the bottom of the gel occasionally. When isolated the 1200 BP product can be ligature into a new vector and cloned on antibiotic selection plates. The resulting colonies produce a plasmid that contains ~200-400 BP of the desired insert but the rest of the insert is a random sequence.
I appreciate any thoughts you all may have and please let me know if you need any more information.
Thanks.
Hi Ian, did you test another polymerase in your experiment? How often did you repeat your experiment with this taq? Cheers, Nadine
It seems like you may be having unspecific amplification and the faint smearing at the bottom of the gel could be your dNTPs, so maybe it would help to use a fresh batch during the next PCR. Also, "bad" dNTPs can lead to unspecific amplification or none at all.
You could also try doing a PCR cycle where you anneal at 62 degrees for 10-15 cycles, and then gradually increase the temperature by 0.2 -0.5 degrees per cycle. If you only get a faint band, it could mean that your annealing temperature is not optimal. Increasing your extension time to 1.5 minutes could also help in getting the full sequence.
Did you sequence the plasmid prior to doing a PCR? If it has been stored for a long time at -20 your plasmid may have degraded.
Best! Diahann
I'd like to know this too... If Q5 can't do it with reasonable tweaking, is there really any other reasonable hope? Has anyone ever found a polymerase that can do stuff that Q5 can't?
From your very good description of the problem I would say the main problem is that your primers are not very specific. The general design appears to be fine, but you should pay attention at their 3' end, where polymerization occurs. My suggestion is a design with the last two nucleotides being the pair CC or GG (the same on both primers to avoid primer dimer formation), but in general in the last 5 nt you should have from 3 to 4 G/C nt, obtaining a dG of -12 to -15 for the 3' end of the primer. This will ensure best chances of specific annealing, allowing you to use high annealing temperatures as you are already using. The problem is that a long primer will give you a high annealing temperature, but this might be too high to keep stably annealed the endings, reducing your PCR efficiency. You can still try your primers at lower annealing temperature, 58 °C, for example. But since you already produce unspecific amplification, I would try increasing the annealing TIME to 45-60 sec. Some primers have slow binding kinetics and increasing the time you give them to bind will considerably increase the specific vs the unspecific annealing.
I would also reduce the primer concentration. On a plasmid it will be sufficient to have 0.25 uM concentration, 1 uM is appropriate for genomic DNA, where your template representation is very low.
Finally, to amplify 2.2 kb with a proof-reading taq polymerase, 20 sec. of extension is a bit too short (that's possibly why you don't go over 1.2 kb of amplification), I think 1.5 min as suggested by Diahann is more appropriate.
Good luck.
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