No PCR product after bisulfite treatment :( - (Jul/27/2005 )

Quick question,

I just finished running my bisulfite treated DNA samples through a thermal cycler with C->T specific primers. I'm pretty sure my primer sets are good (at my home computer or I would post them), but I just tried to run my main product (1244 BP) on a gel and got no visible product. This is going to be a nested PCR reaction so I have my primers set up to amplify.

Main Primer set: 1244 BP
Nested Primer 1: ~400 BP
Nested Primer 2: ~420 BP
Nested Primer 3: ~423 BP

Is it normal to have no product after first round of amplification due to low amounts of DNA (started with 10 ng pre-bisulfite treatment) or what not? I was planning on running my first nested primer set tomorrow but the fact that I have no main primer set product is troubling me. I used a bisulfite treatment regimen fairly similar to labtechies version with embedding DNA into agarose and treated for a full 4 hours. My agarose bead did not seem to melt completely so I'm not sure if that might possibly be the issue. Any ideas?

My PCR conditions were as follows

94C initial denaturation 10 Min

5 Cycles of the following:
Denaturation: 30S @ 95C
Annealing: 90S @ 57C (2C below Tm)
Amplfication: 120S @ 72C

30 Cycles of the following
Denaturation: 30S @ 95C
Annealing: 60S @ 57C (2C below Tm)
Amplification 90S @ 72C

Final amplification of 10 min @ 72C
Hold @ 4C

CW

Hi cwong,

I don't usually see a product after the first round of PCR amplification, the product is there but not abundant enough to be seen as a band on a gel, I hope the second round will give some nice results.

10ng of starting DNA for bisulfite treatment is really stretching the limit of the assay I have to say.

Nick

Hi Nick,

Oops sorry that was supposed to be 100 ng not 10 lol.. sorry. I'll see how the nested PCR goes tomorrow and hopefully it works. Thanks again.

CW

Good luck, I have my fingers crossed for you

Nick