PCR product too short - (Feb/28/2008 )
i´m at the beginning of a cloning experiment. therefore i have designed 2 primer-pairs. in an initial PCR step i tried to amplifiy my target (cDNA source). in 3 of the 4 products i got one band, but with wrong size (smaller than expected) at agarose-gel. and 1 product was only a smear. what was the problem?
i think for the most of you this is easy to answer.
any suggestion could be helpful.
Is this product GC rich? what annealing temperature did you try. How small is the product compared to actual size?
Have you check the primers? Perhaps it is the wrong annealing temperature? Try to run gradient PCR. Cheers..
length of the product is around 3300bp. GC content between 40-50%. i only tried an general annealing temp of 55°C for my first experiment.
additional info see attachment
thanks for help
Your PCR product is too big to amplify in normal PCR, you might test different brand Taq for long PCR products.
By the way, your primer contain short annealing sequence to your target seqeunce, you should using primer containging at least 20 bp annealing sequence to your targets, especially for big PCR product
The portion of your primers which binds to your template is too short. It's 14-16 bp long, when it should be 18-22 bp long. To get good amplification for a somewhat long product, use either a Pfu/Taq mixture or a Pfu like enzyme, rather than straight Taq. My favorites are Invitrogen PCR Supermix High Fidelity, and Phusion premix. The premix eliminates a lot of variability and makes the pipetting easier and more under control. The Tm of your primers should be calculated on the part which binds your template, not on the whole primer. Given your results, I think there is no option but to redesign the primers and try again. Cycling conditions won't rescue these primers.
thanks to all for replies and suggestions, especially to phage.
i use Platium Taq Polymerase High Fidelity. which could be used for synthesis of large fragments (12-20kb).
ok. length of hybridzation is a bit short, i think i will order another 2 pairs and try again?