I am trying to amplify nearly 200bp segment of DNA. Changed primer concentrations, template concentration, primer stock everything, still no amplification. Seeking help.
Yes I tried with different Taq polymerases we had. But nothing worked. The primers which worked previously are not working now. I made new working stock and tried but no amplification!!!
Hello Shivansh,
As Terry suggested you can try new polymerase and buffer. Also if you give as the master mix details and the product company name it will be easy to trouble shoot.
I used the following concentrations in a 20 microlitre reaction:
Taq Pol 0.5
FP 1
RP 1
dNTP 0.5
Taq Buffer 5
Template nearly 50ng (Company: NEB)
are your original primer stocks dissolved in water? If so I think that it has become acidic with co2 absorption from the air and depurinated your primers. This might account for why they used to work but no longer do. Order new primers and dissolve in TE at alkaline pH. the edta chelates divalent ions so nucleases which might be present and need magnesium to work are inactivated. Another possibility is that the primer stocks are stored at the back of a self defrosting freezer and have freeze thawed too many times and have become broken up
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