AlanFung:LabNotes/DNA/2009-3-26: Difference between revisions
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Place column in a 1.5mL tube | Place column in a 1.5mL tube | ||
Add in | Add in 10uL of M-Elution Buffer directly to the column matix | ||
Repeat Centrifuge | Repeat Centrifuge | ||
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Revision as of 17:45, 27 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 gDNA
2uL 100ng/uL gDNA 18uL H20 130uL CT Conversion Reagent
PCR Tube B 0.6ng gDNA
6uL 0.1ng/uL gDNA 14uL H2O 130uL Ct converison Reagent
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
Place column in a 1.5mL tube Add in 10uL of M-Elution Buffer directly to the column matix Repeat Centrifuge
No | sample | sample concentration | 10xLigase buffer | template+cpg30k(50.7ng/ul)+suppressor(8nM each)vol+H2O |
positive control | 200ng/ul | 1ul | 1+1+1+6ul | |
negative control | 0 | 1ul | 1+1+1+6ul |
- 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
x12 Template 10ul 2X iProof Mastermix 50ul 600ul AmpF6.2SoL (10uM) 4ul 48ul AmpR6.2SoL (10uM) 4ul 48ul 50X SYBG I 0.8ul 9.6ul H2O 31.2ul 374.4ul
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 AA AB AC BA BB BC Ladder Sample 0 10 10 10 10 10 10 3 6X Loading Dye 0 2 2 2 2 2 2 3 ----------------------------------------------------------------- Total 14uL 9uL