i am getting a low PCR product ! I have tried reducing the annealing temp, increasing the primer and DNA template conc. too. But still quite low. Anything I should try ?
There can be many contributing factors for getting a low PCR product. You can troubleshoot the following:
1) Check whether the primers you designed are perfect complementary to the DNA strands. Perhaps they are not specific which is one of the major reasons for low PCR product yield. Moreover they should not be self complementary or each others complement. If you designed them manually, I would suggest using a good primer design software. There are many tools available in the market. I personally prefer and use Primer Premier to design my primers and the tool has not disappointed me so far. Here is its link which I found on their website: http://www.premierbi...sign/index.html
2) You can try to reduce the volume of sample in the reaction mix if required and carry out another ethanol precipitation with the samples.
3) Try to optimize Mg2+ ion concentration and use a good proofreading enzyme with high fidelity.
All the best for your assays.