I have tried to run PCR for the vectors under teh following conditions:
Step 1: 95°C for 2 min
Step 2: 95°C for 1 min
Step 3: 55°C for 1 min
Step 4: 68°C for 2 min
Step 5: Cycle to Step 2 25 more times
Step 6: 4°C for infinity
Step 7: End
The PCr product seems to be very less, I get a smiley on the gel. But under the same conditions and same oligos, I got heaps of insert. I am planning to change the annealing temp and the extension time.
In addition, I see smears on my gel at the bottom, could these be from the dNTPs? And how do I get rid of the smears? I am planning to use Dpn1 digestion.
Thanks :)
Hi. You can decrese the anaeling temperature. This causes the high concentration of expected band.furthermore, again design your primers with higher specifity.
Hi
what you can do is to put less primers and dNTP, always in excess even after the PCR (and that's what you see on gels), try a faster protocol (15/15/30 cycles is sufficient), but first analyze your primers by an electronic PCR (as NCBI tool: https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome). you'll see by this tool if your primers are suitable for right amplification and have an idea on the Tm to use.
fred
Hi Sayeeda, these might be not related to the plasmid template but the Gel/Running Buffers - make sure these solutions are not too old and have the correct final concentration. Good luck!
Thank you everyone for your answers.
I would like to mention what I have come up to. \
1. So I was using pfu polymerase which is not so efficient when it comes to bigger fragments. pfu polymerase is usually a good polymerase for short fragments (guess ~ max 3 kb) but for bigger templates it is too slow (2min/kb!). For bigger fragments I I am trying to use Q5 (20-30 sec/kb) polymerase from NEB and hopefully it will work (as it did for some neighbouring labs).
2. And also the Annealing Temperature for the primers is a HUGE change-maker. It MUST be kept optimum for the primers to work best.
Cheers
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