PCR product too short - (Nov/29/2010 )
I'm trying to PCR cDNA from a purified plasmid, but the product I'm getting is only around 200bp when it should be 1.2kb. I've done a BLAST alignment of my primers with the full insert sequence and there's only one binding site for each of them, which should give me the full size product. I'm currently doing 30 cycles, with 2 minute extension and a Tm of 50, using GoTaq polymerase. Any suggestions would be greatly appreciated. Thanks.
Have you looked for binding site on the plasmid itself? Is 50 the optimal annealing temperature for these primers? How much DNA are you adding?
I initially used an annealing temperature of 55, but didn't see any product at all, although that might have been due to having too high a concentration of dNTPs, so I can try increasing the temperature again. I'm using 2ul of my purified plasmid. I haven't been able to check the sequence of plasmid yet, as I'm waiting for the person who gave it to us to get back to me with a map so I can check the size of the insert by restriction digest.
I'm guessing you have WAY too much plasmid in your PCR. Try diluting 100x and 1000x and giving it another try.
2 ul of plasmid is unhelpful -it could be anywhere from 1 ng/ul to 2000ng/ul, which would give very different answers to your problem. What is the concentration (ng/ul) or the ng amount you are adding?
To set up the PCR properly you should be doing annealing temperature and magnesium gradients to determine the optimal conditions. You should also only use a tiny anount of DNA for plasmid based reactions. 10 ng is heaps.
You might try adding DMSO to your PCR master mix, I had similar issues when trying to PCR from a plasmid.
Adding DMSO at 8% of the final volume of rxn helped produce strong bands at the desired size for me, without DMSO i was getting non specific binding everywhere and nothing at the size i was after. And wish i had found out about it before trying to tweak all the salt concentrations/dNTPs etc.
Hope this helps. (Wear gloves as im told DMSO can burn on contact with skin)
Thanks for the help all. Turns out the 'standard' primer concentration is too high for this particular reaction, so it's all working now.