AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 2: Difference between revisions

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==Reagent Preparation==
==Reagent Preparation==
===Proteinase K===
 
*Add 260ul(D5020) or 1040ul(D5021) of proteinase K storage buffer to the tube containing proteinase K
 
*Dissolve completely and store at -20C
===CT Conversion Reagent (1250ul Total good for 9rxns)===
===CT Conversion Reagent (1250ul Total good for 9rxns)===
*CT Conversion Reagent is light sensitive, so minize its exposure to light
*CT Conversion Reagent is light sensitive, so minize its exposure to light
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==For optimal results use 500ng of DNA per treatment (3 treatments for each sample)==
==For optimal results use 500ng of DNA per treatment (3 treatments for each sample)==
===Section II Bisulfite conversion of DNA===
===Section II Bisulfite conversion of DNA===
*Add 200ul of sample to 130ul of CT conversion reagent solution in a pcr tube
{| {{table}}
| Sample||Volume||CT Conversion Reagent||Treatment
|-
| NA12878||20.0||130.0||x3
|-
| 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
*Vortex the sample to mix
*Pulse centrifuge  
*Pulse centrifuge  

Latest revision as of 21:12, 30 December 2009

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

Overview[edit]

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

Sample[edit]

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[edit]

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[edit]

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

  • 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[edit]

  • 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[edit]

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

Section II Bisulfite conversion of DNA[edit]

Sample Volume CT Conversion Reagent Treatment
NA12878 20.0 130.0 x3
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