BSP cycles and primers - Is it OK to use the same primer set but with more (Aug/01/2005 )

Hi, Nick
My BSP has a very weak band after first round of 40cycles, then I run another 40cycles using 1ul of PCR product with the same primer set as the 1st round, then I got a very smart band. Do you think it is OK or this will make any artifact? Thank you very much.

Hi Haiyan,

It is OK. The amplification should be specific. To make sure, always include negative controls (using water as template). Sometimes we have to run 2x40 cycles to get enough products for sequencing.

Reamplifying with the same primers and nested primers works equally well.

I agree with Pcrman! That is a good result Haiyan!

Thank you very much Nick and PCRman.

I have another question: In my gene the CpG island is from the end of the promoter to exon2, do you think I need to check the entire promoter region (2kb) or just stick to the CpG island is enough? Thanks.

Yes, only stick to the CpG island region.

Quick question about sequencing primers.

When designing sequencing primers, is it safe to make the assumption that non-cpg island cpgs will be converted to tpgs? I normally try not to include CG sequences in my sequencing primers but I'm having trouble bumping up my GC content and Tm above 30%/60C so I'm thinking of including 1 CG site, is this alright?

Is the random frequency of methylation outside of islands high enough that I might run into an issue where the site might be methylated causing my designed primer to not bind?

Would I need to use a degenerate/wobble base substitution to take this possibility into account ie: Y sub for C/T or would the single base mismatch in the middle of the sequence still allow the primer to bind?

Cwong

hi cwong,

are you trying to sequence a PCR amplicon? if so I would suggest you clone into a sequecning vector and use degenerate SP6 or T7 primers for sequencing as sequencing of PCR amplicons of BSP are notorisouly difficult to optimise.

If you were to perform direct seqeuncing then sequence using the reverse primer, I am not too sure why but you are more likely to get good chromatogram signal with the reverse primer and never with the forward.

good luck

Nick