I am trying to amplify a gene for a mouse histone linker protein using PCR for cloning purposes. For this, I have used cDNA synthesized from RNA isolated from mouse liver as template, but I am unable to detect any bands after running the PCR samples on an agarose gel. Could anyone please share a suitable protocol for using cDNA as template for PCR cloning? What amount of cDNA should I use per PCR reaction?
Use 500ng-1μg of template. Check the CG content of your template in silico to see if you need a 98°C melting temperature. Use an annealing temperature 5°C lower than the lower primer annealing temperature and have an extension time of 1 min per kb of expected amplicon. If you are not seeing bands after 40 cycles you may need to design new primers.
I will suggest you confirm the potency of the cDNA by running a house keeping gene primer on your PCR, If you get a band you can start the recommended troubleshooting process of the Histone linker protein. I'd also suggest you run an Insilico PCR to have an idea of optimum pcr profile for the Histone linker primers.
I normally use cDNA material of 1ugas template for synthesis. You can do two things here.
1. You can see if the reaction mixture is working for an housekeeping gene, to make sure that the master mix and the cDNA are working fine.
2. You can change the PCR conditions and number of cycles. You basically will have to check the GC content, the size of the amplicon and the salt adjusted Tm of the primers that you are using. If the GC content is very high try denaturation temperatures of 98 degrees. And use extension time of 1min for 1kb.
If by all means you are unable to get a band then study the literature to see whether the gene can be absent in your sample.
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