4 replies to this topic

[epicrazy]

I am optimising a robust set of bisulphite primers. I did an initial gradient between 55 and 62C and found 58 C to be optimal. However I find one non-specific band which varies in intensity between runs. My PCR mixture contains: 0.1mM dNTP final concentration and 1.5mM Mgcl2. My PCR programme was: 15 mins at 95 (hot start taq), 40 cycles of 95C -45 sec, 58C - 1 min, 72C - 1 min.
I am currently trying a touchdown PCR with the following conditions:
95-15 mins
Phase1 : 95 - 30 sec, 65C (with 0.5 decrements per repeat) - 30sec, 72C -30s = 15 cycles
Phase2 : 95 - 30 sec, 58C - 30 sec, 72C - 30s = 15 cycles

Does this programme sound logical? This is the first time Im doing a touchdown PCR. Also, is the ratio of dNTP versus Mgcl2 too low thereby increasing free mgcl2? I have tried 0.2mM but it didnt make a difference.

Please help!

[methylnick]

what is the size of your unexpected band?

[epicrazy]

My band of interest is 212 bp and the non specific products are about 160-180bp... I finally managed to reduce the non specific but never got rid of it completely... since i was worried about getting spurious bands in my digestion, i have finally resorted to gel extraction (i run it on a 2.5%) , but its so tiresome for many samples!!!

[methylnick]

hmmmm, maybe your primers are not as robust as you think, or they are mispriming to other sequences within the genome!!

[epicrazy]

When I do a touchdown, I dont see anything in the first round of PCR products when I run it on a gel. I use the first round products as template and do another 15 cycles to get more product (at a higher temperature to reduce non-specificity). (Done numerous optimisations with first round and second round, but could never eliminate non specific) Because the CpG island is so CG dense, I found it difficult in the first place to design primers (the software didnt pick up any!), so instead of doing a nested, i stuck to the same primer set. So then I run the second round of PCR products on a 2.5% and gel extract quite tedious, but at least its clean.. Im going to sequence it just to be sure that Im amplifying the right region...