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

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Objective[edit]

  • 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[edit]

  • Bisulfite Conversion of DNA
  • Capturing Reaction

Reagent Preparation[edit]

 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[edit]

Bilsulfite Conversion with EZ DNA Methylation-Gold Kit[edit]

  • 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[edit]

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[edit]

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[edit]

  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[edit]

                           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.

  • Ms DNA taken out one cycle earlier

TBE Gel Run[edit]

  • 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

File:ZhangLab 2 2009-06-09 14hr 58min crop.jpg

  • Ms DNA seems to be over amplified
  • Rerun another PCR amplification for Ms DNA template only (-1 cycle)
                           TubeA
  Ms DNA Template          5ul     
  2X iProof Mastermix      50ul     
  AmpF6.3SoL (10uM)        4ul     
  AmpR6.3SoL (10uM)        4ul        
  50X SYBG I               0.4ul   
  H2O                      36.6ul  

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

File:ZhangLab 2 2009-06-09 17hr 28min-2 crop.jpg

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