AlanFung:LabNotes/DNA/2009-3-26

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Revision as of 14:46, 27 March 2009 by >Alan6017518 (→‎Procedures)
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DNA Methylation Capturing - Jurkat gDNA equivalent to 100 cells' DNA

Objective

  • Specific capturing of bisulfite converted genomic targets

Samples & Materials

  • Jurkat gDNA
  • EZ DNA Methylation Direct Kit 03/11/09
  • 10X Ligase Buffer
  • Cpg30k
  • Suppressor
  • RNAse Free H2O
  • dNTP NEB10 mM
  • AmpliTaq Stoffel Fragment
  • Jurkat gDNA (100ug/mL)
  • Dilute 1uL stock gDNA with 99uL RNAse-free H20
  • 2% Agarose Gel

Overview

  • Cell Preparation
  • Bisulfite Conversion
  • Elution
  • Capturing

Procedures

  • Diltue Jurkat gDNA to equal amount of gDNA from 100 cells

100cells(600pg=0.6ng) Dilute 10uL stock gDNA(1ng/uL) with 90uL RNAse free H2O

  • Bisulfite Conversion of DNA

Add in 130uL of CT conversion Reagent Solution directly to the samples

Vortex and spin down

Perform reaction in thermocycler

      Step1   98C, 8m
      Step2   64C, 3.5hr
      Step4   4C,  storage for up to 20 hr

Add 600uL of M-Bindin Buffer into a IC Column

Load 150uL of samples into IC column

CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES

Centrifuge at 20,000g for 30s Discard flow through

Add 100uL M-Wash Buffer to column Repeat Centrifuge

Add 200uL of M-Desulphonation Buffer to column let stand at RT for 20m Repeat centrifuge step

Add 200uL of M-Wash Buffer to the column Repeat Centrifuge

Place column in a 1.5mL tube Add in 8uL of M-Elution Buffer directly to the column matix Repeat Centrifuge

No sample sample concentration 10xLigase buffer template+cpg30k(50.7ng/ul)+suppressor(8nM each)vol+H2O
positive control 200ng/ul 1ul 1+1+1+6ul
negative control 0 1ul 1+1+1+6ul
  • Thermocycler
      Step1   95C, 10m
      Step2   55C, 16hr
      Step3   Add 1uL SLN mix 
      Step4   55C, 4hr
      Step5   95C, 1m
      Step6   55C, 4hr
      Step7   Go back to step 5 repeat 8 time
      Step8   95C, 1m
      Step9   37C, 1m
      Step10  Add 2uL Exonuclease I/III mix
      Step11  37C, 2hr
      Step12  94C, 5m
      Step4   4C,  0m
  • PCR
                                      x12
   Template                10ul      
   2X iProof Mastermix     50ul      600ul
   AmpF6.2SoL (10uM)        4ul       48ul
   AmpR6.2SoL (10uM)        4ul       48ul   
   50X SYBG I             0.8ul      9.6ul
   H2O                   31.2ul    374.4ul


      Step1   98C, 30s
      Step2   98C, 10s
      Step3   58C, 20s
      Step4   72C, 20s
      Step5   Go back to step 2 repeat 7 times
      Step6   98C, 10s
      Step7   72C, 20s
      Step8   Go back to step 6 repeat 9 times
      Step9   72C, 5m
      Step10  15C, 0m
  • Gel Electrophoresis
                                 Gel 1
Well             1     2     3     4     5    6     7      8
-----------------------------------------------------------------
Content          Blank AA    AB    AC    BA   BB    BC     Ladder
Sample           0     10    10    10    10   10    10     3
6X Loading Dye   0     2     2     2     2    2     2      3
-----------------------------------------------------------------
Total                                               14uL   9uL

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