Dinh:Protocols/Capturing Nov13: Difference between revisions

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|Jurkat gDNA - 203.9 ng/ul / converted gDNA (200ng/ul)/ 11-13 converted gDNA (400ng/ul)/ 11-13 converted gDNA (135ng/ul)
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|0.5ul
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=C. Capturing Protocol CpG30K=
=C. Capturing Protocol CpG30K=

Revision as of 22:14, 23 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. 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
Jurkat gDNA - 203.9 ng/ul / converted gDNA (200ng/ul)/ 11-13 converted gDNA (400ng/ul)/ 11-13 converted gDNA (135ng/ul) 0.5ul
Total 40ul

C. Capturing Protocol CpG30K

Positive control: Jurkat converted DNA Negative control: 1ul RNAse free ddH20