In practice long primers SSR have an increased tendency to hybridize to a template despite mismatches making them, in fact, less specific. Generally, best specificity is achieved with primers of 18-25 bases with rich GC content at the 5'-prime end and low at the 3'prime end. The number of cycle also cause this case.

Thanks for answering!

I think I don't have problem with the primers since they have been used a number of times for different studies. Part of my study is to try to work those primers on Philippine varieties of garlic. My issue is more on the variation of the amplified product length between hot start and non-hot start pcr. Also, I forgot to mention that I'm doing hot-start MANUALLY (adding the MgCl2 after the initial denaturation step). The time it takes for me to add the MgCl2 to 30 samples is around 10 min. Does that affect the result of my PCR, hence the different amp product? Thanks!

Not sure if this helps, but could you use MyTaq as your master mix to avoid adding reagents manually?

I am actually planning to use a HS polymerase. However, due to funds limitation, I'm forced to do a manual HS for optimization. I have to make sure that the result of the initial HS procedure is the right one before I go on with the rest of my samples using an HS polymerase.

Sorry I cannot help further except to say I totally understand the limitation on funds!

Thanks anyway :)

I'm really stuck with the study as of the moment since I don't know how to proceed anymore (whether I'll carry on with the HS+TD or just go back to plain TD). I hope anyone out there could somehow enlighten me with the problem.

Further details: My expected product ranges from 114-300 bp, and I do get that when I'm doing the plain TD pcr. However, whenever I do the HS+TD, the amplified product length increases to around 500bp to 1kb. Also, for TD, I usually get monomorphic bands, but in HS+TD the bands are extremely polymorphic. I'm really bummed out with the results because I can't seem to get it right (or at least I don't know which one is correct). Again, hopefully anyone can help with this. Any answer will be appreciated. TIA!

Hi Grace,

Since the expected product is from 114-300 bp, I would use the plain TD PCR if I have to choose one PCR method. It is hard to tell the reasons why the HS+TD gave you larger bands, unless you want to sequence both PCR products and compare them to find out what is going on. By the way, I always set up my PCR on ice (place PCR tubes in a bucket of ice to minimize the possibility of unspecific primer annealing) then perform manually hot-start. Also, you may ask around and borrow a small unit of HS polymerase just to test from your department or different department if you have a restrained budget.

Good luck,

I forgot to mention: some companies also provide FREE samples for researchers to test. For example, samples for 5 PCRs. You may call these companies up and check out.

Best,