AlanFung:LabNotes/DNA/2009-3-26: Difference between revisions

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>Alan6017518
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Revision as of 21:34, 30 March 2009

DNA Methylation Capturing - Jurkat gDNA equivalent to 100 cells' DNA

Objective

  • Specific capturing of bisulfite converted genomic targets

Samples & Materials

  • Jurkat gDNA (100ng/uL)
  • 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

  • Bisulfite Conversion
  • Elution
  • Capturing

Procedures

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

100cells equivalent to 600pg or 0.6ng gDNA


10ng/uL

  Dilute 10uL stock gDNA(100ng/uL) with 90uL RNAse free H2O

1ng/uL

  Dilute 10uL stock gDNA(10ng/uL) with 90uL RNAse free H2O

0.1ng/uL

  Dilute 10uL stock gDNA(1ng/uL) with 90uL RNAse free H2O


  • Bisulfite Conversion of DNA

PCR Tube A - 200ng Jurkat gDNA

           2uL   100ng/uL gDNA          
           18uL  H20                                  
           130uL CT Conversion Reagent       
    

PCR Tube B 0.6ng Jurkat gDNA

           6uL   0.1ng/uL gDNA
           14uL  H2O
           130uL CT converison Reagent

Vortex and spin down

Perform reaction in thermocycler

  • Run>Alan>METH
      Step1   98C, 8m
      Step2   64C, 3.5hr
      Step4   4C,  storage for up to 20 hr


Add 700uL of M-Bindin Buffer into a IC Column (Increase from 600uL, should increase yield)

Load 150uL of samples into IC column

CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES

Centrifuge at 20,000 rcf 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 10uL of M-Elution Buffer directly to the column matix Repeat Centrifuge CPG30K Dilution

  • Diltue CPG30K

0.7ng/uL

  Dilute 10uL stock gDNA(7ng/uL) with 90uL RNAse free H2O

0.07ng/uL

  Dilute 10uL stock gDNA(0.7ng/uL) with 90uL RNAse free H2O


Tube Content Volume A
Template 10uL
10X Ligase Buffer 1uL
7ng/uL CPG30K 7.24uL
RNAse free H2O 1.76uL
Mineral Oil 20uL


Tube Content Volume
B Template 10uL
10X Ligase Buffer 1uL
0.07ng/uL CPG30K 4.29uL
RNAse free H2O 4.71uL
Mineral Oil 20uL
  • Thermocycler
  • Run>Alan>CAP
      Step1  95C, 10m
      Step2  55C, hold at least 16 hrs
      *Add 1uL SLN mix 
      Step3  55C, 4hr
      Step4  95C, 1m
      Step5  55C, 4hr
      Step6  Go back to step 5 repeat 8 time
      Step7  95C, 1m
      Step8  37C, 1m
      *Add 2uL Exonuclease I/III mix
      Step9  37C, 2hr
      Step10 94C, 5m
      Step11 4C,  hold


  • PCR
                            A+B         
   Template                10ul      
   2X iProof Mastermix     50ul      
   AmpF6.2SoL (10uM)        4ul       
   AmpR6.2SoL (10uM)        4ul       
   50X SYBG I             0.8ul      
   H2O                   31.2ul    


      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                                     Ladder
Sample           
6X Loading Dye   
-----------------------------------------------------------------
Total

Results

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