I ran a PCR for my cphA gene consisting 6 same samples but only one sample was amplified and could be seen the bands after running an agarose gel electrophoresis, although the bands weren't that strong.
Those samples were amplified at the same temperature and were treated with the same pipetting instructions (incl. the same primers). I am not sure whether the problem was the sequences of the primers that couldn't recognize the template or probably something elses.
The template that I used was p5k cphA 6308.
Dear David
there are many different reason because a pcr cannot work.
to help you we need some more information regarding your pcr cicle ( temperatures and lenght of the steps) primers theoretical melting temperature, amount of the template that you use and size if the fragment that you would like to amplify.
Manuele
I agree totally with Manuele Martinelli that there is not enough detail and too many reasons for failure but while you give more details try doing the reaction at 4C lower annealing temperature and see what happens
Dear my friend
- It seem you are using different template. If it is, I want to know, you use a complete master include component for amplifying (master mix), water and primer or you add primer to each template tube separately? Maybe one of your primers is absent here.
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