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

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='''DNA Methylation Capturing - 100 GM20431 cells'''=  
='''DNA Methylation Capturing - Jurkat gDNA equivalent to 100 cells' DNA '''=  




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==Samples & Materials==
==Samples & Materials==
*Jurkat gDNA
*Jurkat gDNA (100ng/uL)
*EZ DNA Methylation Direct Kit 03/11/09
*EZ DNA Methylation Direct Kit 03/11/09
*10X Ligase Buffer
*10X Ligase Buffer
*Cpg30k
*Cpg30k
*Suppressor
*SLN Mix
AmpliTaq Stoffel fragment  2U/ul      2uL
AmpLigase                  0.5U/ul    1uL
Ligase Buffer              10X        1uL
dNTP                      10nM        20uL     
ddH2O                                  5.8uL
 
*RNAse Free H2O
*RNAse Free H2O
*dNTP NEB10 mM
*dNTP NEB10 mM
Line 21: Line 27:


==Overview==
==Overview==
*Cell Preparation
*Bisulfite Conversion
*Bisulfite Conversion
*Elution
*Elution
Line 28: Line 33:
==Procedures==
==Procedures==


Incubate the samples at 50C for 20 mins
* 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
*Bisulfite Conversion of DNA
Add in 130uL of CT conversion Reagent Solution directly to the digested samples
 
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
Vortex and spin down


Perform reaction in thermocycler
Perform reaction in thermocycler
 
*Run>Alan>METH
       Step1  98C, 8m
       Step1  98C, 8m
       Step2  64C, 3.5hr
       Step2  64C, 3.5hr
       Step4  4C,  storage for up to 20 hr
       Step4  4C,  storage for up to 20 hr


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


Load 150uL of samples into IC column
Load 150uL of samples into IC column
Line 47: Line 74:
'''CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES'''
'''CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES'''


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


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Add 200uL of M-Wash Buffer to the column
Add 200uL of M-Wash Buffer to the column
Repeat Centrifuge
Repeat Centrifuge
[REPEAT WASHING STIP FOR ONE SET ONLY]


Place column in a 1.5mL tube
Place column in a 1.5mL tube
Add in 8uL of M-Elution Buffer directly to the column matix
Add in 10uL of M-Elution Buffer directly to the column matix
Repeat Centrifuge
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
*Setup the Capture Reaction
{| border="1" align="center"
|rowspan="6" align="center"|Tube A
| align="center" style="background:#f0f0f0;"|'''Content '''
| align="center" style="background:#f0f0f0;"|'''Volume '''
|-
|Template||align="right"|10uL
|-
|10X Ligase Buffer||align="right"|1uL
|-
|7ng/uL CPG30K||align="right"|7.24uL
|-
|RNAse free H2O||align="right"|1.76uL
|-
|Mineral Oil||align="right"|20uL
|-
|Total Volume
| colspan="2" align="right"| 40uL
|}
{| border="1" align="center"
|rowspan="6" align="center"|Tube B
| align="center" style="background:#f0f0f0;"|'''Content '''
| align="center" style="background:#f0f0f0;"|'''Volume '''
|-
||Template||align="right"|10uL
|-
||10X Ligase Buffer||align="right"|1uL
|-
||0.07ng/uL CPG30K||align="right"|4.29uL
|-
||RNAse free H2O||align="right"|4.71uL
|-
||Mineral Oil||align="right"|20uL
|-
|Total Volume
| colspan="2" align="right"| 40uL
|}


*Thermocycler
*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 4 repeat 8 times
      Step7  95C, 1m
      Step8  37C, 1m
      *Add 2uL Exonuclease I/III mix
      Step9  37C, 2hr
      Step10 94C, 5m
      Step11 4C,  hold


      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
*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
       Step1  98C, 30s
Line 98: Line 188:
  Well            1    2    3    4    5    6    7      8
  Well            1    2    3    4    5    6    7      8
  -----------------------------------------------------------------
  -----------------------------------------------------------------
  Content          Blank AA    AB    AC    BA  BB    BC    Ladder
  Content          Blank                                     Ladder
  Sample          0    10    10    10    10  10    10    3
  Sample           
  6X Loading Dye  0    2    2    2    2    2    2      3
  6X Loading Dye   
  -----------------------------------------------------------------
  -----------------------------------------------------------------
  Total                                               14uL  9uL
  Total
 
==Results==
 


==Suggestion==
==Suggestion==

Latest revision as of 22:40, 31 March 2009

DNA Methylation Capturing - Jurkat gDNA equivalent to 100 cells' DNA [edit]

Objective[edit]

  • Specific capturing of bisulfite converted genomic targets

Samples & Materials[edit]

  • Jurkat gDNA (100ng/uL)
  • EZ DNA Methylation Direct Kit 03/11/09
  • 10X Ligase Buffer
  • Cpg30k
  • SLN Mix
AmpliTaq Stoffel fragment  2U/ul       2uL
AmpLigase                  0.5U/ul     1uL
Ligase Buffer              10X         1uL
dNTP                       10nM        20uL       
ddH2O                                  5.8uL
  • 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[edit]

  • Bisulfite Conversion
  • Elution
  • Capturing

Procedures[edit]

  • 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
  • Setup the Capture Reaction
Tube A Content Volume
Template 10uL
10X Ligase Buffer 1uL
7ng/uL CPG30K 7.24uL
RNAse free H2O 1.76uL
Mineral Oil 20uL
Total Volume 40uL


Tube B Content Volume
Template 10uL
10X Ligase Buffer 1uL
0.07ng/uL CPG30K 4.29uL
RNAse free H2O 4.71uL
Mineral Oil 20uL
Total Volume 40uL
  • 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 4 repeat 8 times
      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

Suggestion[edit]