How stable are PCR-amplicons from bisulfite-treatment? - (Mar/26/2007 )
Hi everybody,
I amplified (BSP-PCR) successfully after bisulfite treatment (Qiagen Epitect-Kit and Zymo EZ DNA Methylation Gold) 540 bp amplicons with a nested PCR. I checked my products after the second PCR-run on a 2% agarose-gel; clear bands were visible.
After that I tried to re-amplify the PCR-products from the run 1 from the antecedent nested PCR and I got only smeared, unspecific bands on my agarose -gel! 
All PCR´s were set up on the same day, the products from the nested PCR were kept frozen until my attempt of reamplification.
It is possible that amplicons degrade completely so fast ? 
Second question: I noted that every time that I check my products on an agarose-gel, beside the amplicon-bands there are always some unspecific bands (primer-dimers, products from the carrier-RNA (QIAGEN-kit)?) on a low range (maybe 40-80 bp long).
a. can this products be eliminated with a PCR-purification kit?
b. if not, does these products interfere in sequencing?
Thanks for your help,
Tharom
Tharom,
like normal, pcr amplicons, bisulfite amplicons are robust and will not degrade so quickly.
smears indicate non-specific amplification and you may need to increase your Tm in your PCR to remove this. In doing so you will reduce your primer dimer and RNA contaminations in question 2.
Nick
Just curious, what is the purpose of studying methylation of PCR amplicons in the first place? Use them as negative control?
no, studying methylation pattern of the amplified promotor region versus gene -expression.
I just was wondering that i was not able to reamplify the products from the first PCR-run a second time with nested primer. The first time it amplifyed well with nested pr., the second time, 5 hours later, no amplification from that probe with that same pr.
After nested PCR I want to sequence my amplicons to see their methylation pattern.
have I understand right your question?
Tharom
a. can this products be eliminated with a PCR-purification kit?
b. if not, does these products interfere in sequencing?
Hi Tharom,
in some cases I wasn't able to get rid of these products by playing with PCR conditions. Using a normal PCR purification kit did not help, amplicons were only poorly sequencable. But I started to purify by gel-extraction, cutting out only the desired band and that allways did the trick.
For the other question - if this happend to me I would guess that I spoiled the pippeting.. forgot the primer, used the wrong template ...
my first round bsp amplicons are well after several weeks at -20°C...Krümel
Hi Krümelmonster,
I understand, ..and how much sample did you purify with gel-extraction (which concentration did you have after purification)? I need 100 ng DNA for sequencing. I amplified with a nested pcr system, i think the conc. must be high enough..? (Bands on gel (8ul sample) were always good visible).
Thanks for your help
Tharom
Hi Tharom,
I normally use 20µl of PCR reaction and purify them with the Qiaquick System. Afterwards I determine the amount of DNA using a small DNA mass ladder. The yields are always sufficient for direct cycle sequencing (up to 500ng).
K.