Dinh:Protocols/Capturing Nov13: Difference between revisions
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Reaction Mix (1x, 150ul): | Reaction Mix (1x, 150ul): | ||
{| border="1" | |||
!Reagent | |||
!Final Conc. | |||
!Vol (1x) | |||
|- | |||
|ddH20 | |||
| | |||
|10ul | |||
|- | |||
|Jurkat gDNA (203.9ng/ul) | |||
| 2.039ug per tube | |||
|10ul | |||
|- | |||
|Conversion Reagent (prepared 11/6) | |||
| | |||
|130ul | |||
|} | |||
Reaction Program: | Reaction Program: |
Revision as of 22:19, 23 November 2009
A. Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold Kit
Reaction Mix (1x, 150ul):
Reagent | Final Conc. | Vol (1x) |
---|---|---|
ddH20 | 10ul | |
Jurkat gDNA (203.9ng/ul) | 2.039ug per tube | 10ul |
Conversion Reagent (prepared 11/6) | 130ul |
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. PCR Amplification of Converted DNA
- Primers - From IDT
-------------------------------------------------- 0.1_F_chr22_31384238GTGAATAGGTTAAGTGAGGTAGAAG 0.1_R_chr22_31384238AAAAAAATCAAACACCAACTATAAA 0.8_F_chr21_39672131AAAATATTGGGATTATAGGTATGAGT 0.8_R_chr21_39672131AACTTCTAAACTAACCAAAACAAAA 0.9_F_chr8_119031762TTATAGTTTGGGTGATAGAGTAAGATT 0.9_R_chr8_119031762AAACCCTAAACAAAATACTCAATATAA --------------------------------------------------
Reagent | Final Conc. | Vol (1x) |
---|---|---|
ddH20 | 7.5ul | |
NEB Tag 2x Master Mix | 1x | 20ul |
Forward Primer (Chr8/21/22) (3.3uM) | ~0.5uM | 6ul |
Reverse Primer (Chr8/21/22) (3.3uM) | ~0.5uM | 6ul |
A/B/C/D | 0.5ul | |
Total | 40ul |
A. Jurkat gDNA - 203.9 ng/ul B. converted gDNA by Alan (200ng/ul) C. 11-13 converted gDNA (400ng/ul) D. 11-13 converted gDNA (135ng/ul)
C. Capturing Protocol CpG30K
Positive control: Jurkat converted DNA Negative control: 1ul RNAse free ddH20