AlanFung:Protocol/Capturing: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
No edit summary
Line 1: Line 1:
==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
==Overview==
==Overview==
*Bisulfite Conversion of DNA
*Bisulfite Conversion of DNA
*Capturing Reaction
*Capturing Reaction


 
===Calculation===
 
==Procedures==
  Probe:target ratio 200:1  
  Probe:target ratio 200:1  
   
   
  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 therefore we use 4uL of gDNA template for bisulfite conversion
 
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
 
==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 therefore we use 4uL of gDNA template for conversion


*One genome mass = 3pg
*One genome mass = 3pg
Line 87: Line 27:
  # of probes needed/ concentration of probes = Volume of probes needed
  # 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
  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==
==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:
RNA
==Capturing System Setup==
==Capturing System Setup==
                        
                        
Line 94: Line 52:
   H2O                              8.17ul
   H2O                              8.17ul
   Total                            20ul  
   Total                            20ul  
  95C 2min -> 60C 24h  
95c 10min -> 60C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 4h -> <BR>4 cycels (95C 1min -> 55C for  4h),add 2ul more SLN mix after 1st cycle -> 95C 5min -> <BR>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
    -> 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

Revision as of 21:53, 4 June 2009

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

Overview

  • Bisulfite Conversion of DNA
  • Capturing Reaction

Calculation

Probe:target ratio 200:1 

NIH_3T3_gDNA Template(100ng/uL)
Use 200ng total, so = 2uL
  • 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

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:

RNA

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 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