texasepigenetics

Yes, I was under the impression her results were "good," however it seems that needing performing the PCR 10 plus times before you get a band at the proper size does not constitute a good bisulfite-PCR Oops!

An update for anyone interested: after working with someone with lots more methylation analysis than myself, it seems the primers I was using are poorly designed. I'll be redesigning following my experienced friend's guidelines and will update if I have any success with what I did for the redesign.

Sorry, I should have corrected the annealing temp- I performed a gradient PCR between 60 and 50- the annealing temp for the primers that did work on the methylated DNA ended up being around 54*C.

I purchased a new stock of primers, not using her old ones.

I am also thinking that it may be as you suggested- that M.SssI is causing additional denaturing or fragmentation. Maybe I'll throw my DNA into the bioruptor for a quick round of sonication before my next trial...
I should also mention that this region is a CpG island- is it also possible that because the region is hypomethylated, that there are so many uracils in the sequence following conversion that it is degrading?

Finally, anyone have insight into using water rather than the elution buffer provided with the zymo gold methylation kit?

Thanks!!

Hi pcrman, thanks for the reply!

I am using the same PCR protocol as the other member- however she used a different thermocycler the Ta at which my methylated DNA produced a band was a few degrees higher than what she says she produced a band at using those primers. The PCR master mix I use has a aptamer inhibitor, TQ21-11. I think I've seen it discussed in these forums, also. I am using a basic non-proofreading taq polymerase.

I am doing two rounds:

PCR1 mix recipe:
MM2/TQ21 mastermix 5x    20%
Taq polymerase 5U/ul          1%
Mix at room temperature. Let the oligo bind to the enzyme.
Water                                  77%
Mix, transfer on ice and keep on ice from now on.
Primer forward 10 uM          1%    final 0.1 uM
Primer reverse 10 uM           1%

Aliquot in PCR tubes or wells (18 ul per well). Keep on ice.
Add bisulfite-treated DNA 2 uL
Heat up the PCR block to 95C.
Transfer the PCR tubes or plate from ice to the hot block.
Start cycling:
Initial denaturation 95C 5 minutes.
Then cycle denaturation 94C 15 seconds, annealing/extension 60 seconds at 60C. Use 40 cycles.
If your annealing temperature needs to be below 60C, add an extension step of 72C for 10 seconds. (mine are, so I added it)

PCR2 mix recipe:
MM2 mastermix 5x                       20%
Taq polymerase 5U/ul                   1%
Water                                            77%
Mix, transfer on ice and keep on ice from now on.
Primer forward (nested)10 uM        1%    final 0.1 uM
Primer reverse  10 uM                     1%

Aliquot in PCR tubes or wells (24.5 ul per well). Keep on ice.
Add 0.5uL PCR1.

Heat up the PCR block to 95C.
Transfer the PCR tubes or plate from ice to the hot block.
Start cycling:
Initial denaturation 95C 5 minutes.
Then cycle denaturation 94C 15 seconds, annealing/extension 60 seconds at 60C. Use 40 cycles.
If your annealing temperature needs to be below 60C, add an extension step of 72C for 10 seconds (mine are, so I added it).

For recovery of my DNA, I am using the columns provided with the zymo kit, but have been eluting with water instead of the included elution buffer- could that be the problem? I don't think I've ever eluted with an elution buffer that comes with columns, in case the components inhibit downstream reactions...