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

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>Alan6017518
(New page: Draft EZ_DNA_Methylation_Direct_Kit_Protocol PDF ==Overview== *Sample preparation *Sample Digestion with Proteinase K *Bisulfite Conv...)
 
>Alan6017518
 
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Draft
==Bisulfite Conversion of Sample NA12878,NA18507, NA20431, CV-Fibr, CV-iPS-B, CV-iPS-F w/+ve and -ve Control==
 
[[Media:EZ_DNA_Methylation_Direct_Kit_Protocol.pdf|EZ_DNA_Methylation_Direct_Kit_Protocol PDF]]


==Overview==
==Overview==
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*Sample Digestion with Proteinase K
*Sample Digestion with Proteinase K
*Bisulfite Conversion of DNA
*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==
{| {{table}}
| 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==
==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===
*CT Conversion Reagent is light sensitive, so minize its exposure to light
*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
*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 by frequent vortexing at RT for 10m
*Mix 160ul of M-Reaction Buffer and mix an additional 1m
*Mix 160ul of M-Reaction Buffer and mix an additional 1m
===M-Wash Buffer===
===M-Wash Buffer===
*Add 24ml of 100% ethanol to the 6ml M-Wash Buffer concentrate (D5020)before use
*Add 24ml of 100% ethanol to the 6ml M-Wash Buffer concentrate (D5020)before use
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==Protocol==
==Protocol==
==Sample Preparation==


==For optimal results use 8x10^4 cells per treatment==
 
*Turn on heat bath to 50C before sample prep
==For optimal results use 500ng of DNA per treatment (3 treatments for each sample)==
*Wash cells with PBS once and perform a cell count
===Section II Bisulfite conversion of DNA===
*Sample Digestion with Proteinase K
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|
| Sample||Volume||CT Conversion Reagent||Treatment
| align="center" style="background:#f0f0f0;"|'''A-2000 cells'''
|-
| align="center" style="background:#f0f0f0;"|'''B-100,000 cells'''
| NA12878||20.0||130.0||x3
|-
| NA18507||20.0||130.0||x3
|-
| NA20431||20.0||130.0||x3
|-
|-
| M-Digestion Buffer (2X)||10ul||13ul
| CV-Fibr||20.0||130.0||x3
|-
|-
| Sample||up to 9ul||up to 12ul
| CV-iPS-B||20.0||130.0||x3
|-
|-
| Proteinase K||1ul||1ul
| CV-iPS-F||20.0||130.0||x3
|-
|-
| H20||X ul||x ul
| Jurkat||20.0||130.0||x1
|-
|-
| Total Volume||20ul||26 ul
| Blank (ddH2O)||20.0||130.0||x1
|-
|-
|  
|  
|}
|}
*Incubate sample(s) for 20m at 50C
*Add 20ul of sample to 130ul of CT conversion reagent solution in a pcr tube
*If following procedure A, procedd directly to Section II
*If following procedure B, mix content throughly with vortex then centrifuge for 5m at 10,000g
*Extract 20ul of the supernatant for bisulfite conversion
 
===Section II Bisulfite conversion of DNA===
*Add 200ul 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