HOW to explain my MSP result of negative control? - (Jul/13/2006 )
peripheral lymphocyte DNA modified with sodium bisulfite was employed as negative control in my research,but this negative control show amplification with both unmethylated- and methylated-primer sets, Not only with unmethylated- primer sets. WHY??? who can help me?
(PCR volume 25 ul ,anealing T 62, 32-35cycles,)
rockysofar,
there are a number of possibilities, one could be that you have a mixed population of lymphocyte cells that contain the locus in both methylation states, another could be that your number of cycles maybe a bit much, try reducing to 15-20. You didn't mention what the calculated Tm of your M and U primer sets were, if they were close to the Tm used in your PCR that should be fine.
N
there are a number of possibilities, one could be that you have a mixed population of lymphocyte cells that contain the locus in both methylation states, another could be that your number of cycles maybe a bit much, try reducing to 15-20. You didn't mention what the calculated Tm of your M and U primer sets were, if they were close to the Tm used in your PCR that should be fine.
N
my anealing T was about 2 degree higher than the calculated Tm.I already tried to reduce the cycle number,but when the cycle was reduced to 30,both of the M and U pcr products were not seen in the gel.
How can I settle this problem?
I have the same problem.But I use sssI do with my sample as positive control.And I found bands with U primers.I don't know how to do that too.Please help me!I am depressed.I spent too much time in this.
Lisa
Lisa
Are you sure your SssI-modification was completely succesful?! I've always used more units in the reaction than recommended by the supplier. Chemicon offers completey methylated and unmethylated DNA.
jörg
Thank you,jörg(a strange name)
That may be the problem.I doubt my SssI-modification is not complete.I just followed their recommendation . Could you please tell me how much SssI is enough for 1 ug DNA?
I bought the positive control from Chemicon before,but that is human DNA,and I use DNA from mice.
Again,Do you have any idea for promoter methylation microarray?
Have a good weekend!
Lisa
Can not anybody help me?Where is pcrman and methylnick?I am very anxious and need an answer.Thanks in advance!
Lisa
I ran into a similar issue and ended up doing 3 consecutive treatments with the SSI enzyme (with phenol chloroform extraction between each) to ensure full conversion.
lla,
a good way to check if your DNA is methylated would be to restriction digest with methylsensitive enzymes such as HpaII or HhaI, this will give you a good indication of the success of your SssI methylase, if it is incomplete, the chances are that your SAM is depleted from the reaction and adding more, or like cancergeek suggests multiple treatments, would ensure this.
good luck!
ps: apologies for not chasing this up for you. 
Thanks,thanks,thanks.......my good friends and savior.
Lisa