I am doing multiplex with 5 products. I am getting some unspecific banding and could use some advice on additives.
I have tried DMSO at 0% 2% 5% and 10% with significant inhibition at the 5% and 10% concentrations and no real help with the unspecific bands.
This is my first PCR optimization and do not know the benefits/downfalls to other additives such as formamide and BSA.
My primers are short 18-19bp due to the very close relatedness of the 5 species I am probing for and this made it nearly impossible to make species specific primers with longer sequences. My optimal Tm is 50 degrees for my multiplex reaction after runing trials with gradient PCR, thus can not use higher Tm to remove these unspecific bands without loosing 3/5 of my bands.
I was successful in amplifying every species with my designed primers and they were all specific in singleplex, but with over 1000 predators to screen, multiplex will make my life immensly easier.
Is formamide or BSA worth testing? Is there anything else I can try?
Here is my reaction w/o any DMSO ect.
10uL 10x Buffer
0.2uL Taq Polymerase
19.8uL PCR water
1uL of each for/rev primers X5 species =10uL (25ng/uL)
1uL template for each species x5- 5uL (25ng/uL)
Total 50 uL
I can't control for template concentration when I do my real experiment so modifying template isn't possible.