AlanFung:Protocol/Capturing: Difference between revisions
>Alan6017518 |
>Alan6017518 |
||
Line 94: | Line 94: | ||
H2O 8.17ul | H2O 8.17ul | ||
Total 20ul | Total 20ul | ||
95C 2min | 95C 2min -> 60C 24h | ||
-> add 1ul SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) | -> add 1ul SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) | ||
-> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul Exo I&III | -> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul Exo I&III | ||
-> 37C 2h -> 90C 5min -> 4C hold | -> 37C 2h -> 90C 5min -> 4C hold |
Revision as of 01:28, 4 June 2009
Overview
- Bisulfite Conversion of DNA
- Capturing Reaction
Procedures
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 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
- 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
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 -> 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