Troubleshooting with Bisulfite - (Mar/01/2007 )
does anyone knows what happens if the BS solution is too concentrated? (6-7M instead of 5)
I have tryed out some protocols with certain data and didn't check the calculations before. The PCR has failed, but it worked with the positive controls so I think the PCR conditions are ok, the trouble must be with the BS treatment. On the other hand, can anyone suggest a checkpoint og the succesfullness of BS treatment just _before_ the PCR?
Thanks for any advice!
i always use a supersaturated solution of bisulfite.
the concentrations you state would suggest that not all the bisulfite powder went into solution. This is fine.
as for a checkpoint, I am afraid PCR is the best way. If you don''t have amplification of your samples, maybe because you have lost most of your template in the process. This is normal because up to 90% of your DNA is degraded by bisulfite.
I may suggest you perform a second round or third round of PCR and see if you can get something.
Yes, that's true, I usually need to filtrate my BS solution before apply it to the beads. So you don't think that this could be a problem... in an agarose bead, what is the lowest amount of DNA which can be treated and amplified? I don't want to make so big pools... I need to work with small amount of starting material.
shouldn't be a problem. I would say, and you wouldn't need to filter your solution.
if you are working with low amounts of starting material, you may consider using a kit for conversion, MethylEasy is the only one that claims no degradation of DNA so you don't lose anything, and it seems to work quite well.
Just want to find out if bisulfite treatment is harsher on the unmethylated residues as opposed to the methylated ones.
I have ran the MSP for my samples and it's always the unmethylated primed sets that do not give me a good band despite its corresponding positive control giving me a good one.
So I have eliminated the PCR reaction and also looking at troubleshooting the bisulfite treatment.
Anyone have any idea??
Thanks in advance!