I trying to amplify N40 ssDNA library with hot start polymerase, but template was not amplified.
I tried different combination of cycle number, annealing temperature and time of extension and annealing.
Can anyone give me suggestions?
Hi Smriti,
you should first test the quality of your primers doing an in-silico PCR as in the UCSC website (http://genome.ucsc.edu/cgi-bin/hgPcr) or at NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) just to see if your primers are abble to amplify the targeted region.
fred
Hi Smriti,
you should first test the quality of your primers doing an in-silico PCR as in the UCSC website (http://genome.ucsc.edu/cgi-bin/hgPcr) or at NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) just to see if your primers are abble to amplify the targeted region.
fred
Smiriti
- there could be a whole host of reasons including primer related issues; so start with Frederic's suggestion
- Provide your exact method in an attached file and then people will be better able to comment: This is complex and details might illuminate the problem
Another issue you could check is quality and quantity of your extracted DNA
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