AlanFung:LabNotes/DNA/2009-3-26
DNA Methylation Capturing - 100 GM20431 cells
Objective
- Specific capturing of bisulfite converted genomic targets
Samples & Materials
- Jurkat gDNA
- EZ DNA Methylation Direct Kit 03/11/09
- 10X Ligase Buffer
- Cpg30k
- Suppressor
- RNAse Free H2O
- dNTP NEB10 mM
- AmpliTaq Stoffel Fragment
- Jurkat gDNA (100ug/mL)
- Dilute 1uL stock gDNA with 99uL RNAse-free H20
- 2% Agarose Gel
Overview
- Cell Preparation
- Bisulfite Conversion
- Elution
- Capturing
Procedures
- Turn on the incubator heat it up to 50C
- Cell Preparation
Resuspended cells in T25 flask by repeat pipetting
Perform cell counting
- Cell Count of GM20431 P20: 207,656 cells/mL
To get 1,000,000 cells: 1,000,000/(207,656cells/mL)=4.816mL
aspirate 4mL + 816uL cell suspension and transfer to a 50mL centrifuge tube
Spin down at 10,000 rpm for 5 mins
Aspirate supernatant completely
Resuspended cells with 1000uL UV treated PBS (1000cells/uL)
- Cell Dilution
Performed cell dilution to reach concentration of 11.11 cells/uL and 1.11 cells/uL
- 111.11 cells/uL
Dilute 10uL (1000cells/uL)cell suspension with 80uL UV TREATED PBS IN PCR TUBE
- 11.11 cells/uL
Dilute 10uL (111.11 cells/uL) cell suspension with 90 uL UV TREATED PBS IN PCR TUBE
- 1.11 cells/uL
Dilute 10uL (11.11 cells/uL) cell suspension with 90 uL UV TREATED PBS IN PCR TUBE
- Sample Digestion with Proteinase K
1rxn x 6 -------------------------------------------- RNAse-free H2O 0.0 0.0 Protinase K 1.0 1.0 M-Digestion Buffer (2X) 10.0 10.0 11.11 cells/uL 9.0 -------------------------------------------- 20uL 20uL
Vortex and spindown
Incubate the samples at 50C for 20 mins
- Bisulfite Conversion of DNA
Add in 130uL of CT conversion Reagent Solution directly to the digested samples
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
- Thermocycler
Step1 95C, 10m Step2 55C, 16hr Step3 Add 1uL SLN mix Step4 55C, 4hr Step5 95C, 1m Step6 55C, 4hr Step7 Go back to step 5 repeat 8 time Step8 95C, 1m Step9 37C, 1m Step10 Add 2uL Exonuclease I/III mix Step11 37C, 2hr Step12 94C, 5m Step4 4C, 0m
- PCR