I have been trying to PCR amplify an ~3kb fragment from cDNA of THP1 macrophage to put restriction sites at both the ends so that I can use it for restriction cloning. My primers are 36bp long with 24 annealed bases, 6bp of the RD site, and 6bp for the enzyme to sit; they have a Tm of ~53°C with a GC content of 44.4% and 41.7%. I set up a gradient PCR from 47C-59C and didn't get a single band of good in any of the lanes (image attached). I eluted my specific 3kb band (gave an inferior yield of 4-8ng/ul) and used it as a template to put another PCR, expecting to see just one band (since, now, only my desired template should've been there taking isoforms also out) but still saw a similar pattern (as seen in the attached image) of smaller non-specific amplicons.

What can I do to have no non-specific amplicons either (so that I can proceed with PCR cleanup instead of gel elution to prevent yield loss) or increase the intensity of my specific band?

More Tanmay Devgan's questions See All
Similar questions and discussions