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AlanFung:Protocol/EZ DNA Methylation-Direct
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Draft [[Media:EZ_DNA_Methylation_Direct_Kit_Protocol.pdf|EZ_DNA_Methylation_Direct_Kit_Protocol PDF]] ==Overview== *Sample preparation *Sample Digestion with Proteinase K *Bisulfite Conversion of DNA ==Reagent Preparation== ===Proteinase K=== *Add 260ul(D5020) or 1040ul(D5021) of proteinase K storage buffer to the tube containing proteinase K *Dissolve completely and store at -20C ===CT Conversion Reagent=== *CT Conversion Reagent is light sensitive, so minize its exposure to light *Add 790ul M-solubilization buffer and 300ul M-Dilution Buffer to a tube of CT Conversion Reagent *Mix by frequent vortexing at RT for 10m *Mix 160ul of M-Reaction Buffer and mix an additional 1m ===M-Wash Buffer=== *Add 24ml of 100% ethanol to the 6ml M-Wash Buffer concentrate (D5020)before use *Add 96ml 100% ethanol to the 24ml M-Wash buffer concentrate (D5021) before use ==Protocol== ==Sample Preparation== ==For optimal results use 8x10^4 cells per treatment== *Turn on heat bath to 50C before sample prep *Wash cells with PBS once and perform a cell count *Sample Digestion with Proteinase K {| {{table}} | align="center" style="background:#f0f0f0;"| | align="center" style="background:#f0f0f0;"|'''A-2000 cells''' | align="center" style="background:#f0f0f0;"|'''B-100,000 cells''' |- | M-Digestion Buffer (2X)||10ul||13ul |- | Sample||up to 9ul||up to 12ul |- | Proteinase K||1ul||1ul |- | H20||X ul||x ul |- | Total Volume||20ul||26 ul |- | |} *Incubate sample(s) for 20m at 50C *If following procedure A, procedd directly to Section II *If following procedure B, mix content throughly with vortex then centrifuge for 5m at 10,000g *Extract 20ul of the supernatant for bisulfite conversion ===Section II Bisulfite conversion of DNA=== *Add 20ul of sample to 130ul of CT conversion reagent solution in a pcr tube *Vortex the sample to mix *Pulse centrifuge *Perform *98C for 8m *64C for 3.5hr *4C hold *Add 600ul of M binding buffer into a column assembly *Load sample(s) to the column *Close the cap and mix by inverting the column several times *Centrifuge at >10,000g for 30sec *Discard the flow through *Add 100ul of M-Wash Buffer to the column *Centrifuge at full speed for 30sec *Add 200ul of M-Desulphonation buffer to the column and let stand at RT for 20m *Centrifuge for 30sec *Add 200ul of M-Wash buffer to the column and repeat centrifuge *Place the column into a 1.5ml tube *Add 10ul of M-elution buffer directly to the column matrix (Volume can be adjusted, depending on the requirements) *Centrifuge for 30sec to elute the DNA
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