What are the conditions that need to be optimised for PCR amplification of 1237 bp DNA fragment for cloning of VEGF whole protein. I used different annealing temperatures and periods of time. but the max. size I got is 830 bp?
Dear Mohammed
which polimerase and which extention time are you using?
1237bp is not so big also the standard TAq polimerase can be able to amplify it ! It seems more that you have some problem in primer specificity or in the template?
Which template are you using?
ciao
Manuele
Your problem is probably primer specificity, in which case you can try to use higher annealing temperatures. Make sure you also use the right amount of DNA template (as recommended by your polymerase supplier). Finally, you can play with the extension time based on the speed of your polymerase (ex : 1min30 for a 1kb/min polymerase).
If you keep getting a 830 bp amplicon instead of 1237 bp, you may need to redesign primers.
Make sure you double-check primer specificity through primer-blast
https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi
I absolutely agreed with Marting Chenal's answer. Please check the primer and redesign it if needed. Based on the type of the polymerase you are using, try to increase the extension time like to 1.5 min for 1237bp. Good luck!
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