I did PCR reaction without using any PCR kit . The result is not good enough. I got heavy low non specific band and probably primer dimer (picture attached). First, I did with annealing temperature of 55. After plasmid isolation I got the good band and the PRC was done using that plasmid DNA. I permed this with 35 cycle. Could you please help me to overcome this issues?
I think that this is primer dimer. Use less primer 1/2, 1/4 and 1/8 of your current amount. Run an annealing temperature gradient if you have a good cycler. Either use a hot start Taq enzyme or do a manual hot start by heating your sample and taq mastermix to 90c then adding the primer mix and start cycling immediately. Check your primers correctly align on the sequence and try amplifying a range of template amounts in case your template is contaminated.
Once you haveany primer dimer it amplifies much better than template so the important thing is to stop p-d and give the specific amplification a chance to work
I think that this is primer dimer. Use less primer 1/2, 1/4 and 1/8 of your current amount. Run an annealing temperature gradient if you have a good cycler. Either use a hot start Taq enzyme or do a manual hot start by heating your sample and taq mastermix to 90c then adding the primer mix and start cycling immediately. Check your primers correctly align on the sequence and try amplifying a range of template amounts in case your template is contaminated.
Once you haveany primer dimer it amplifies much better than template so the important thing is to stop p-d and give the specific amplification a chance to work
You should not need to do 35 cycles with a plasmid template, 20 or 25 is ample. But in addition to all the good suggestions that Paul Rutland made, be sure to run a control reaction to make sure (hopefully one came with the kit) to be sure all the components are working correctly. And I agree to be sure and double check your primers are correct, I've seen many instances where someone mistakenly makes a primer from the wrong strand or with reversed sequence.
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