Hi Nawshin,

When I test new primers, I make six serial 1:10 dilutions starting at 10ng of DNA. I'm doing a slightly different application (using genomic DNA instead of cDNA), but the same approach should work. Don't forget to do a melting curve in addition to checking the PCR efficiency. Good luck!

Dear Nawshin

concur with Nathaniel. A 1:10 serial dilution of your cDNA starting with 1ul of neat cDNA (approximating to 10ng as stated) down to 1:10 000

Alternatively for low copy number genes a 1:2 serial dilution beginning with neat cDNA and progressing to 1:128 sometimes in my experience works better for this subset of GOI than the aforementioned log(10) dilution

Good luck !!

Hi Nawshin,

               I make two serials of Working standards (10:90 and 1:99). Try it