No band on BSP - (Jul/28/2005 )

I treated the DNA according to the procotol on-line(http://www.protocol-online.org/prot/Detailed/3160.html). With modification by using (Promega A7280) to purify nucelic acid at step 8; Without using TE(PH, 7.5), but using the TE(PH8.0) . After the treatment, I run a gel to find out find DNA degraded into smears which suggested treatment might have in effective.

So I started PCR according to the conditions provied by the methprimer for BSP. After the PCR, I found there are some smears without a clear bind.

Does anybody can help me in checking the primer?
Besides primer, are there any other suggestions?

can you post the primers to the board mou1? And also the gel image of your DNA.

I don't normally see any gDNA after bisulfite treatment.

we can then have a look and see if the primers are okay.

Nick

For people doing bisulfite PCR I recommend reading this paper:

Bisulfite genomic sequencing: systematic investigation of critical experimental parameters by C. Grunau, S. J. Clark and A. Rosenthal.

The full-text is freely available at http://nar.oxfordjournals.org/cgi/content/full/29/13/e65

According to this paper, 84–96% of DNA is degraded during the modification reaction. Running a gel using the treated DNA doesn't tell anything.

please check the attachment.

hi mou1, these primers are not ideal for BSP, methoprimer doesn't pick them too correctly.

attached is where I would pick my primers. Again going on pcrman's cited paper within it, there are certain criteria for primer selection of BSP primers, methprimer does a good job selecting MSP primers though.

The primer seqeunces are underlined in white. check the calculated Tm for them and make sure they are equivalent, if they are not, adjust the length by including or excluding bases from the 5' end.

good luck!

Nick

Hi, Nick,

I choose the primer with the shading. The total length of both primers is 25, GC content is 28%, and the Tm is 60 by [2(a+t)+4(g+c)].

Compared the two primers, what's the reason make the second pair perfect?

Shall I devise another pair for nest PCR?

Hi mou,

basic parameters for primer design, is to pick ones with a large nujmber of conversion events (about 30% of the primer contain T's which were C's prior to bisulfite treatment).

Furthermore, your primer should end in a conversion event or two or three, you should take the G off the 3' end of the reverse primer.

Fully nested sets of primers are ideal, but in terms of cost, it's just as good to design a hemi-nested set (ie: 3)

Good luck!!

Nick

Hi Nick,

May I use the the underlined sequence for the nested primer in combing the forward primer? Thank you.

mou1,

that is okay, but I think the primer (doubled underlined) would be better.......it includes more conversion events than the primer you have selected. There are also quite a few G residues that would help in bumping up the Tm.


good luck!

Nick

Nick,

The new one already has 27 nt. How many nt would be proper for the Primer. Do you think it would be better to add more nt with the yellow shdow?

Thanks.