AlanFung:LabNotes/DNA/2009-3-26: Difference between revisions
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Revision as of 21:36, 30 March 2009
DNA Methylation Capturing - Jurkat gDNA equivalent to 100 cells' DNA
Objective
- Specific capturing of bisulfite converted genomic targets
Samples & Materials
- Jurkat gDNA (100ng/uL)
- 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
- Bisulfite Conversion
- Elution
- Capturing
Procedures
- Diltue Jurkat gDNA to equal amount of gDNA from 100 cells
100cells equivalent to 600pg or 0.6ng gDNA
10ng/uL
Dilute 10uL stock gDNA(100ng/uL) with 90uL RNAse free H2O
1ng/uL
Dilute 10uL stock gDNA(10ng/uL) with 90uL RNAse free H2O
0.1ng/uL
Dilute 10uL stock gDNA(1ng/uL) with 90uL RNAse free H2O
- Bisulfite Conversion of DNA
PCR Tube A - 200ng Jurkat gDNA
2uL 100ng/uL gDNA 18uL H20 130uL CT Conversion Reagent
PCR Tube B 0.6ng Jurkat gDNA
6uL 0.1ng/uL gDNA 14uL H2O 130uL CT converison Reagent
Vortex and spin down
Perform reaction in thermocycler
- Run>Alan>METH
Step1 98C, 8m Step2 64C, 3.5hr Step4 4C, storage for up to 20 hr
Add 700uL of M-Bindin Buffer into a IC Column (Increase from 600uL, should increase yield)
Load 150uL of samples into IC column
CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES
Centrifuge at 20,000 rcf 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
Place column in a 1.5mL tube Add in 10uL of M-Elution Buffer directly to the column matix Repeat Centrifuge CPG30K Dilution
- Diltue CPG30K
0.7ng/uL
Dilute 10uL stock gDNA(7ng/uL) with 90uL RNAse free H2O
0.07ng/uL
Dilute 10uL stock gDNA(0.7ng/uL) with 90uL RNAse free H2O
Tube | Content | Volume | A | Template | 10uL |
10X Ligase Buffer | 1uL | ||||
7ng/uL CPG30K | 7.24uL | ||||
RNAse free H2O | 1.76uL | ||||
Mineral Oil | 20uL |
Tube | Content | Volume |
B | Template | 10uL |
10X Ligase Buffer | 1uL | |
0.07ng/uL CPG30K | 4.29uL | |
RNAse free H2O | 4.71uL | |
Mineral Oil | 20uL |
- Thermocycler
- Run>Alan>CAP
Step1 95C, 10m Step2 55C, hold at least 16 hrs
*Add 1uL SLN mix
Step3 55C, 4hr Step4 95C, 1m Step5 55C, 4hr Step6 Go back to step 5 repeat 8 time Step7 95C, 1m Step8 37C, 1m
*Add 2uL Exonuclease I/III mix
Step9 37C, 2hr Step10 94C, 5m Step11 4C, hold
- PCR
A+B Template 10ul 2X iProof Mastermix 50ul AmpF6.2SoL (10uM) 4ul AmpR6.2SoL (10uM) 4ul 50X SYBG I 0.8ul H2O 31.2ul
Step1 98C, 30s Step2 98C, 10s Step3 58C, 20s Step4 72C, 20s Step5 Go back to step 2 repeat 7 times Step6 98C, 10s Step7 72C, 20s Step8 Go back to step 6 repeat 9 times Step9 72C, 5m Step10 15C, 0m
- Gel Electrophoresis
Gel 1 Well 1 2 3 4 5 6 7 8 ----------------------------------------------------------------- Content Blank Ladder Sample 6X Loading Dye ----------------------------------------------------------------- Total