I was tried to get band for cloning of NLRP3 gene which is of 3000 bp length. For standardization of annealing temperature, I run gradient PCR with combination of different concentration of primers and cDNA from mice tissue. But every time I am getting near to 500 bp and there is no band in negative control (NC). I have added DMSO to the reaction to avoid non-specific amplification, reduced amplification cycles and many other measures which could contribute for smearing. But no improvement in the gel. This is my third set of primers (designed as well as obtained from literature). Please give me some advice. I am attaching picture of gel. Thanks in advance
Did you adjust your elongation time to account for the length of the insert you are trying to amplify? You can find out what speed your polymerase has, and you need to ensure that you give it enough time each cycle to fully replicate your insert.
Michael Adams Yes, elongation time was 3 min as my gene of interest is of 3.1kb length.
You can try to use a specific primer designed by the specific programs for that and then test it. Also, you can try using RAPD primers and the produced bands you can take it as a molecular marker.
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