AlanFung:LabNotes/Capturing/Ms Meth/NIH 3T3 Ms gDNA/2009-6-5: Difference between revisions

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Line 95: Line 95:
   Ms DNA Template          5ul      0uL
   Ms DNA Template          5ul      0uL
   2X iProof Mastermix      50ul    50uL
   2X iProof Mastermix      50ul    50uL
   AmpF6.2SoL (10uM)        4ul      4ul
   AmpF6.3SoL (10uM)        4ul      4ul
   AmpR6.2SoL (10uM)        4ul      4ul   
   AmpR6.3SoL (10uM)        4ul      4ul   
   50X SYBG I              0.4ul    0.4ul
   50X SYBG I              0.4ul    0.4ul
   H2O                      36.6ul  36.6ul
   H2O                      36.6ul  36.6ul


98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.  
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.  
*Purification with Qiaquick column
 
==TBE Gel Run==
*0.5uL low mass ladder + 3uL 6X gel loading dye
*3uL amplified Jurkat DNA + 3uL 6X gel loading dye
*3uL amplified Ms DNA + 3uL 6X gel loading dye
 
*Ms DNA seems to be over amplified
*Rerun another PCR amplification for Ms DNA template only
 
 
*Gel band is more concentrated around 400bp, however there is still a smear, may be due to capturing of varying length of targets.

Revision as of 00:38, 10 June 2009

Objective

  • Prepare two tubes of bisulfite converted Ms and Jurkat gDNA enough for plenty of experiments
  • Perform capturing with MS_Meth probes and Alice's Probes for positive control
  • Follow closely to Ida's protocol


Overview

  • Bisulfite Conversion of DNA
  • Capturing Reaction

Reagent Preparation

 dNTP concentration 10mM(NEB)
                                                                    ddH2O     Ligase Buffer   Ligase     Amplitaq    dNTP           
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP:      0.5ul         0.1ul       0.1ul       0.2ul    (10mM)0.1ul 
                                                           x30       15ul          3ul        3ul        6ul      3ul


Procedures

Bilsulfite Conversion with EZ DNA Methylation-Gold Kit

  • Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent vortex for 10M
  • Add 24mL of 100% ethanol to the 6mL M-Wash Buffer concentrate
  • Add 130ul of CT conversion reagent to samples. If your DNA sample is less than 20uL make up the difference with H2O
  • Mix the sample and perform
  • 98C 10min -> 64C 150min -> 4c overnight.
  • Add 600ul M-Binding buffer to spin columns.
  • Add the converted samples to the columns, close the gap and mix by inverting several times.
  • Spin at 15,000rpm for 30sec, discard flow through
  • Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through.
  • Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec.
  • Add 200ul M-Wash Buffer, spin for 30 sec.
  • Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec.
  • Measure the DNA with Nanodrop:
*Ms_gDNA (CT Converted) 384.7ng/uL * 10uL
*Jurkat_gDNA (CT Converted) 368.7ng/uL * 10uL
*Dilute Ms_gDNA (CT Converted) with 7.85uL ddh2o to 200ng/uL
*Dilute Jurkat_gDNA (CT Converted) with 7.17uL ddh2o to 200ng/uL

Calculation for Ms_Meth Probe Set

Probe:target ratio 200:1 

NIH_3T3_gDNA CT Converted Template(200ng/uL)
Use 200ng total, so = 1uL
  • One genome mass = 3pg
  • First calculate the # of genome your target represents
  • For 200ng, it represents 200ng/3pg=66666.67 genomes
  • You want to have 200x the probes as the target 200ng/3pg*200
  • You want every single probe to target ratio to be 200:1 200ng/3pg*200*55,000
Ms_Meth 22ng/uL 55,000Probes 100nt
Since 1 base = 330Da (for single stranded DNA, 660Da for ds), 
the concentration of probe is 22 ng/ul /(100x330) = 0.000666667 = 666.67nM
# of molecules per mole = 6X10^23
# of probes needed/ concentration of probes = Volume of probes needed
the volume the probe needed is 200ng/3pg x 200 x 55,000 / [(6x10^23)x (666.67*10^-9M)]*10^6 =1.83uL

Calculation for CPG30K probe Set

Probe:target ratio 200:1 

Jurkat_gDNA CT Converted Template(200ng/uL)

Use 200ng total, so = 1uL
  • One genome mass = 3pg
  • First calculate the # of genome your target represents
  • For 200ng, it represents 200ng/3pg=66666.67 genomes
  • You want to have 200x the probes as the target 200ng/3pg*200
  • You want every single probe to target ratio to be 200:1 200ng/3pg*200*30,000
CPG_30K 10ng/uL 30,000Probes 100nt
Since 1 base = 330Da (for single stranded DNA, 660Da for ds), 
the concentration of probe is 10 ng/ul /(108x330) = 0.000280584 = 280.58nM
# of molecules per mole = 6X10^23
# of probes needed/ concentration of probes = Volume of probes needed
the volume the probe needed is 200ng/3pg x 200 x 30,000 / [(6x10^23)x (280.58*10^-9M)]*10^6 =2.37uL

Capturing System Setup

  CT Converted Ms-gDNA (200ng/uL)      1uL            
  10X AmpLigase buffer                 1uL
  Ms_Meth Probe (242.4nM)              1.83uL             
  H2O                                  6.17uL
  Total                                10uL
95c 10min -> 60C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 4h -> 
4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle -> 95C 5min ->
add 2ul Exonuclease I/III mixadd 2ul Exonuclease I/III mix as soon as the temperature is lowered to 37C -> 37C 1h -> 94C 5min -> 4C hold


*Noy enough SLN mix, added less than 2uL for the Jurkat DNA capturing reaction


PCR with AmpF/R6.2Sol and iProof

                           TubeA    TubeB  
  Jurkat DNA Template      0uL      5uL 
  Ms DNA Template          5ul      0uL
  2X iProof Mastermix      50ul     50uL
  AmpF6.3SoL (10uM)        4ul      4ul
  AmpR6.3SoL (10uM)        4ul      4ul   
  50X SYBG I               0.4ul    0.4ul
  H2O                      36.6ul   36.6ul

98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.

TBE Gel Run

  • 0.5uL low mass ladder + 3uL 6X gel loading dye
  • 3uL amplified Jurkat DNA + 3uL 6X gel loading dye
  • 3uL amplified Ms DNA + 3uL 6X gel loading dye
  • Ms DNA seems to be over amplified
  • Rerun another PCR amplification for Ms DNA template only


  • Gel band is more concentrated around 400bp, however there is still a smear, may be due to capturing of varying length of targets.