Hi everyone,
I’m working with primers where the initial binding temperature is around 60-62°C, but with tails, it increases to 69-70°C. Should I use step-up PCR, or is adding DMSO more advisable? Also, I have a very low amount of gene of interest cDNA from a plant sample, where the gene of interest is expressed at very low levels.
So far, my standard gradient PCR(60 to 72) attempts haven’t been successful. Any advice would be greatly appreciated.
Thank you!
Note: I know the true answer is " try both because that is a PCR you know its lika a quantum biology some times" but I wonder which way is better choice for PCR masters
An expensive suggestion is whole genome amplificatin of your dna then you have plenty and can run gradients of temperature and dmso if yur cDNA is high GC.
DMSO behaves like increasing the annealing temperature as far as the primers are concerned so In this case where template is scarce I would just run a sample at Ta 55c and if you get a dirty but positive amplification then you can run a dmso gradient of 0-7% dmso or raise the Ta in 2c steps with or without step up
I have seen situations with overhangs where the step-up made all the difference. There's generally no substitute for trying all the things, and seeing what happens. Most importantly use a modern polymerase (Superfi 2) and try different primer pairs (if you have the freedom to do so).