SarahLee8

Hello,

I will be attempting a PCR and I was wondering how much of my reaction with my first primer set I should add to the second reaction.  I've also read that an exonuclease can be added before the second reaction to eliminate anything that is left of the first primer set.  Is this necessary?

Thank you.

Hello all,

I am wondering if I could get some troubleshooting feedback.

I have been performing bisulfite conversion using the ZymoResearch Methylation Gold kit.  However, after PCR, I am not getting any bands using my BSP primers.  I put .3 ug of DNA into my bisulfite conversion reaction.  Some other feedback I have gotten is that my amplicon is too large for BSP.  My current target is 762 bps and I know that most of the literature recommends between 200-300 bps.  However, I have seen papers with larger amplicons around the size of mine.  Does anyone have any experience with bisulfite treatment and PCR of larger amplicons?  Are there any other troubleshooting recommendations for my protocol?

Please let me know if additional information is needed.

Thank you.

Hello-

I'm considering using the Zymo Methylated & Non-methylated pUC19 DNA Set for use in my bisulfite sequencing experiments, but I'm not entirely sure how it works as an internal control.  Any insight would be very much appreciated!

Thanks

Hello, I'm looking for some help in regards to the design of bisulfite sequencing primers.

As I'm reviewing the literature, I've seen that some papers refer to primer sets designed specificially for either the bottom strand or the top strand.  This may have a simple explanation that I am missing, but I am still having a difficult time grasping this... When we design primers for normal PCR, we pick a primer from the top strand and a primer from the reverse strand.  How would we pick primers from just one strand if the sequences are the same, but just the reverse complement of each other?  I appreciate any insight you can provide!

Thank you.