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

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
Line 12: Line 12:


==Sample Preparation==
==Sample Preparation==
{| {{table}}
| Sample Prep||ng/ul||To Get 1000ng||Make it up to 40ul
|-
| NA12878||160.000||6.250||33.750
|-
| NA18507||257.000||3.891||36.109
|-
| NA20431||314.000||3.185||36.815
|-
| CV-Fibr||712.000||1.404||38.596
|-
| CV-iPS-B||358.000||2.793||37.207
|-
| CV-iPS-F||309.000||3.236||36.764
|-
|
|}


==Reagent Preparation==
==Reagent Preparation==

Revision as of 23:06, 2 December 2009

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 1000ng Make it up to 40ul
NA12878 160.000 6.250 33.750
NA18507 257.000 3.891 36.109
NA20431 314.000 3.185 36.815
CV-Fibr 712.000 1.404 38.596
CV-iPS-B 358.000 2.793 37.207
CV-iPS-F 309.000 3.236 36.764

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

  • 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

Sample Preparation

For optimal results use 500ng of DNA per treatment

  • Turn on heat bath to 50C before sample prep
  • Wash cells with PBS once and perform a cell count
  • Sample Digestion with Proteinase K
A-2000 cells B-100,000 cells
M-Digestion Buffer (2X) 10ul 13ul
Sample up to 9ul up to 12ul
Proteinase K 1ul 1ul
H20 X ul x ul
Total Volume 20ul 26 ul
  • Incubate sample(s) for 20m at 50C
  • 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
  • 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