AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 2

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Bisulfite Conversion of Sample NA12878,NA18507, NA20431, CV-Fibr, CV-iPS-B, CV-iPS-F w/+ve and -ve Control

Overview

  • Sample preparation
  • Sample Digestion with Proteinase K
  • Bisulfite Conversion of DNA

Sample

NA12878 (160ng/ul)
NA18507 (257ng/ul)
NA20431 (314ng/ul)
CV-Fibr (712ng/ul)
CV-iPS-B (358ng/ul)
CV-iPS-F (309ng/ul)

Sample Preparation

Sample Prep (ng/ul) To Get 1500 ng (uL) Make it up to 60ul
NA12878 160.0 9.4 50.6
NA18507 257.0 5.8 54.2
NA20431 314.0 4.8 55.2
CV-Fibr 712.0 2.1 57.9
CV-iPS-B 358.0 4.2 55.8
CV-iPS-F 309.0 4.9 55.1

Reagent Preparation

CT Conversion Reagent (1250ul Total good for 9rxns)

  • 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

For optimal results use 500ng of DNA per treatment (3 treatments for each sample)

Section II Bisulfite conversion of DNA

Sample Volume CT Conversion Reagent Treatment
NA12878 20.0 130.0 x2
NA18507 20.0 130.0 x3
NA20431 20.0 130.0 x3
CV-Fibr 20.0 130.0 x3
CV-iPS-B 20.0 130.0 x3
CV-iPS-F 20.0 130.0 x3
Jurkat 20.0 130.0 x1
Blank (ddH2O) 20.0 130.0 x1
  • 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
  • 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

Qubit Quantificatioin