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==Suggestion== *Taq 2X Mastermix is used instead of iQ supermix -Change to 2X iQ supermix *Annealing temperature 62C form the protocol is too high for the primers -Lower it to 50C (Do not change the condition) *Design primer for the target sequence without being methylated -Run PCR using both set of primers to confirm the effectiveness of methylation *Too much sample(80,000cells) resulting in incomplete bisulfite conversion -Use 10,000 cells next for conversion *PCR Preparation not done on the cooling block, and Zymo recommended using a hot-start ploymerase -Prepare reaction on cooling block and use a hot-start polymerase *Using 2% agarose gel to run gel electrophoresis should get better resolution *Use higher amount of ladder and load ladder next to the sample well.
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