AlanFung:Protocol/Capturing: Difference between revisions

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  NIH_3T3_gDNA Template(100ng/uL)
  NIH_3T3_gDNA Template(100ng/uL)
  Use 200ng total, so = 2uL
  Use 200ng total, so = 2uL we double the amount to compensate the lost during the CT conversion


*One genome mass = 3pg
*One genome mass = 3pg

Revision as of 01:18, 4 June 2009

Overview

  • Bisulfite Conversion of DNA
  • Capturing Reaction

Calculation

Probe:target ratio 200:1 

NIH_3T3_gDNA Template(100ng/uL)
Use 200ng total, so = 2uL we double the amount to compensate the lost during the CT conversion
  • 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

Procedures

Prepare CT Conversion Reagent as follows

1. Add 790 µl of M-Solubilization Buffer and 300 µl of M-Dilution Buffer to a tube of CT Conversion Reagent.

2. Mix at room temperature with frequent vortexing or shaking for 10 minutes.

3. Add 160 µl of M-Reaction Buffer and mix an additional 1 minute.

Note: It is normal to see trace amounts of undissolved reagent in the CT Conversion Reagent. Each tube of CT Conversion Reagent is designed for 10 separate DNA treatments.

Storage: The CT Conversion Reagent is light sensitive, so minimize its exposure to light. For best results, the CT Conversion Reagent should be used immediately following preparation. If not used immediately, the CT Conversion Reagent solution can be stored overnight at room temperature, one week at 4°C, or up to one month at -20°C. Stored CT Conversion Reagent solution must be warmed to 37°C, then vortexed prior to use.

  • Bisulfite Conversion of DNA

Add in 130uL of CT conversion Reagent Solution directly to the 2uL of DNA sample compensate with water in order to make up to 20uL (18uL)

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 [REPEAT WASHING STIP FOR ONE SET ONLY]

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

Measure concentration with nanodrop

Capturing System Setup

  CT Converted Ms-gDNA             8ul     
  10X AmpLigase buffer             2.0ul   
  Ms_Meth Probe (242.4nM)          1.83ul    
  H2O                              8.17ul
  Total                            20ul 
  95C 2min -> -0.02C/sec to 60C -> 60C 24h 
    -> add 1ul SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) 
    -> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul Exo I&III
    -> 37C 2h -> 90C 5min -> 4C hold