Starting with DNA methylation analysis - (Jun/15/2005 )
I "mock" convert my sequences with Microsoft Word and the find and replace tool and look for areas with no CpG's and with a great number of potential converted C's along with the criteria outlined in Warnecke et al 2002 Methods.
I then calculate the Tm and ensure the primer pairs are equivalent aiming for 60C.
order and off I go.
It was not that long ago where primer picking programs were not as accessible as they are now!
As for your primer3 page, there is an option to tell promer3 to exclude selecting primers within a particular region within your seqeunce, default i think there are a number of ranges which you would have to edit before the program will work. A note though, primer3 will not design bisulfite or MSP primers for you, you need to use methprimer which is a modified primer3 for methylation analysis.
I know this is a relatively old topic, but I had a question about the EZ DNA Methylation kit from Zymo. I was thinking of trying this kit out but again, the elution volume worries me slightly. I'm not sure what the recovery rate for this kit based method is, but I'm assuming that a good amount of the DNA is still lost in the conversion process.
Has anyone diluted their eluted samples 1:10 or greater? I'm planning on running a nested PCR reaction, but I'm not sure how dilute is too dilute with this kit.