Dinh:Protocols/Capturing Nov13: Difference between revisions
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>Dinh mNo edit summary |
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Line 12: | Line 12: | ||
4C -> overnight | 4C -> overnight | ||
Purification: | Column Purification: | ||
1. Place a Zymo-Spin IC Column into a provided Collection tube. | 1. Place a Zymo-Spin IC Column into a provided Collection tube. | ||
2. Add 600 ul of M-Binding Buffer to spin column | 2. Add 600 ul of M-Binding Buffer to spin column |
Revision as of 00:54, 14 November 2009
A. Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold Kit
Reaction Mix (1x, 150ul):
Jurkat DNA (203.9ng/ul) - 10ul RNase free ddH20 - 10 ul (DNA = 2.039ug) Conversion Reagent (prepared 11/6) - 130 ul
Reaction Program:
98C -> 10 min 64C -> 2.5 hours 4C -> overnight
Column Purification:
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.
B. Capturing Protocol CpG30K
Positive control: Jurkat converted DNA Negative control: 1ul RNAse free ddH20