Hello all,

I have been having some trouble with our bisulfite converted DNA. I am using Zymo's EZ DNA Methylation-Gold Kit. I've been following their protocol very closely as this is the first time I've worked with bisulfite conversion. The DNA used was extracted from rat hippocampal tissue and will be used for pyro-sequencing.

I put in 500ng of DNA into the initial conversion and checked the RNA concentration after it came off the thermocycler using a NanoDrop. These concentrations are all around 17000ng/ul, which from my understanding means that the conversion process is working well. After the recovery steps is when everything seems to be getting lost. The end concentrations I've been getting are no higher than 20ng/ul, most are not even this high.

I've made sure the DNA going in is of good quality and there isn't too high or too low of a concentration going into the process. If using one of the extended thermocycling options could be avoided that would be ideal. What would be the best way to increase these recovery rates?