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=A. November 13, 2009 Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold Kit= Reaction Mix (1x, 150ul): {| border="1" !Reagent !Final Conc. !#1 (3x) !#2 !#3 |- |ddH20 | |10ul |15ul |19ul |- |Jurkat gDNA (203.9ng/ul) | varies per tube |10ul |5ul |1ul |- |Conversion Reagent (prepared 11/6) | |130ul |130ul |130ul |} Reaction Program: 98C -> 10 min 64C -> 2.5 hours 4C -> overnight Column Purification *Washing was done twice: 1. Place a Zymo-Spin IC Column into a provided Collection tube. 2. Add 600 ul of M-Binding Buffer to spin column 3. Load the reaction mix into the spin column. 4. Close cap and mix by inverting 10 times. Centrifuge at 15K rpm for 30 s. Discard flow through. 5. Add 100ul of M-Wash Buffer. Centrifuge at 15K rpm for 30 s. Discard flow through. 6. Add 200ul M-Desulphonation Buffer and wait for 20 min. Centrifuge at 15K rpm for 30 s. Discard flow through. 7. Add 200ul M-Wash Buffer. Centrifuge at 15K rpm for 30 s. Discard flow through. 8. Add another 200ul M-Wash Buffer. Centrifuge at 15K rpm for 30 s. Discard flow through. 9. Place the column into 1.5ml microcentrifuge tube. Add 12ul M-Elution Buffer into center of column matrix. Wait 1 min. Centrifuge at 15K rpm for 30 s. 10. Measure the DNA with Nanodrop (use M-Elution Buffer to blank) {| border = "1" !Sample # !Input Jurkat gDNA (ng) !Output converted gDNA (ng) ! % yield |- | 1 | 2039ng | 198.8ng/ulx10ul = 1988ng | 97% |- | 2 | 1019.5ng | 79ng/ulx10.5ul = 829.5ng | 81% |- | 3 | 203.9ng | 20ng/ulx9.6ul = 192ng | 94% |} Notes: Next time, repeat using 2x of sample 1 reaction and use 14ul M-Elution buffer to elute first column, then use flow-through to elute second column.
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