I am trying to amplify my genes of interest.Though I got the amplified product of the desired size on my first attempt , i am not able to get the amplification again. What might be the problem and how should i troubleshoot ?
There are umpteen numbers of reasons why a reaction may not yield results you wish. But since you already had it working, start with identifying the specific difference(s) between your first and second experiment.
Trouble-shooting may include not only what goes into your reaction mixture (quality and integrity of enzyme, primers, dNTPs, template etc) but also the tubes and the machine you use... all changes need to be considered.
Hi Bhoomika,
There are a couple of things you can check for troubleshooting: https://www.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide
Are you doing anything anything different from your first PCR that worked? Did you store you DNA template and polymerase at the right conditions? Are you adding enough Mg2+ and primers? These are questions you should ask yourself.
Best of luck with future experiments!
Hey Bhoomika,
Firstly, you should check your template its concentration whether it's same as you have used in your first reaction or re isolated from same source, check its concentration by nano drop. Next, you can check your MgCl2 concentration in your reaction, to improve its specificity and you can also check the annealing temperature to re amplify it.
hope it will work
good luck.
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