AlanFung:LabNotes/DNA/2009-3-26: Difference between revisions
>Alan6017518 |
>Alan6017518 |
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*10X Ligase Buffer | *10X Ligase Buffer | ||
*Cpg30k | *Cpg30k | ||
* | *SLN Mix | ||
AmpliTaq Stoffel fragment 2U/ul 2uL | |||
AmpLigase 0.5U/ul 1uL | |||
Ligase Buffer 10X 1uL | |||
dNTP 10nM 20uL | |||
ddH2O 5.8uL | |||
*RNAse Free H2O | *RNAse Free H2O | ||
*dNTP NEB10 mM | *dNTP NEB10 mM | ||
Line 43: | Line 49: | ||
*Bisulfite Conversion of DNA | *Bisulfite Conversion of DNA | ||
PCR Tube A - 200ng gDNA | PCR Tube A - 200ng Jurkat gDNA | ||
2uL 100ng/uL gDNA | 2uL 100ng/uL gDNA | ||
18uL H20 | 18uL H20 | ||
130uL CT Conversion Reagent | 130uL CT Conversion Reagent | ||
PCR Tube B 0.6ng gDNA | |||
PCR Tube B 0.6ng Jurkat gDNA | |||
6uL 0.1ng/uL gDNA | 6uL 0.1ng/uL gDNA | ||
14uL H2O | 14uL H2O | ||
130uL | 130uL CT converison Reagent | ||
Vortex and spin down | Vortex and spin down | ||
Perform reaction in thermocycler | Perform reaction in thermocycler | ||
*Run>Alan>METH | |||
Step1 98C, 8m | Step1 98C, 8m | ||
Step2 64C, 3.5hr | Step2 64C, 3.5hr | ||
Step4 4C, storage for up to 20 hr | Step4 4C, storage for up to 20 hr | ||
Add | |||
Add 700uL of M-Bindin Buffer into a IC Column (Increase from 600uL, should increase yield) | |||
Load 150uL of samples into IC column | Load 150uL of samples into IC column | ||
Line 66: | Line 74: | ||
'''CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES''' | '''CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES''' | ||
Centrifuge at 20, | Centrifuge at 20,000 rcf for 30s | ||
Discard flow through | Discard flow through | ||
Line 81: | Line 89: | ||
Add in 10uL of M-Elution Buffer directly to the column matix | Add in 10uL of M-Elution Buffer directly to the column matix | ||
Repeat Centrifuge | Repeat Centrifuge | ||
{| border="1" | 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 | |||
*Setup the Capture Reaction | |||
{| border="1" align="center" | |||
|rowspan="6" align="center"|Tube A | |||
| align="center" style="background:#f0f0f0;"|'''Content ''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume ''' | |||
|- | |||
|Template||align="right"|10uL | |||
|- | |||
|10X Ligase Buffer||align="right"|1uL | |||
|- | |||
|7ng/uL CPG30K||align="right"|7.24uL | |||
|- | |||
|RNAse free H2O||align="right"|1.76uL | |||
|- | |||
|Mineral Oil||align="right"|20uL | |||
|- | |||
|Total Volume | |||
| colspan="2" align="right"| 40uL | |||
|} | |||
{| border="1" align="center" | |||
|rowspan="6" align="center"|Tube B | |||
| align="center" style="background:#f0f0f0;"|'''Content ''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume ''' | |||
|- | |- | ||
| | ||Template||align="right"|10uL | ||
| | |||
| align=" | |||
|- | |- | ||
| || | ||10X Ligase Buffer||align="right"|1uL | ||
|- | |- | ||
| || | ||0.07ng/uL CPG30K||align="right"|4.29uL | ||
|- | |||
||RNAse free H2O||align="right"|4.71uL | |||
|- | |||
||Mineral Oil||align="right"|20uL | |||
|- | |||
|Total Volume | |||
| colspan="2" align="right"| 40uL | |||
|} | |} | ||
*Thermocycler | *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 4 repeat 8 times | |||
Step7 95C, 1m | |||
Step8 37C, 1m | |||
*Add 2uL Exonuclease I/III mix | |||
Step9 37C, 2hr | |||
Step10 94C, 5m | |||
Step11 4C, hold | |||
*PCR | *PCR | ||
Line 136: | Line 188: | ||
Well 1 2 3 4 5 6 7 8 | Well 1 2 3 4 5 6 7 8 | ||
----------------------------------------------------------------- | ----------------------------------------------------------------- | ||
Content Blank | Content Blank Ladder | ||
Sample | Sample | ||
6X Loading Dye | 6X Loading Dye | ||
----------------------------------------------------------------- | ----------------------------------------------------------------- | ||
Total | Total | ||
==Suggestion== | ==Suggestion== |
Latest revision as of 22:40, 31 March 2009
DNA Methylation Capturing - Jurkat gDNA equivalent to 100 cells' DNA [edit]
Objective[edit]
- Specific capturing of bisulfite converted genomic targets
Samples & Materials[edit]
- Jurkat gDNA (100ng/uL)
- EZ DNA Methylation Direct Kit 03/11/09
- 10X Ligase Buffer
- Cpg30k
- SLN Mix
AmpliTaq Stoffel fragment 2U/ul 2uL AmpLigase 0.5U/ul 1uL Ligase Buffer 10X 1uL dNTP 10nM 20uL ddH2O 5.8uL
- 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[edit]
- Bisulfite Conversion
- Elution
- Capturing
Procedures[edit]
- 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
- Setup the Capture Reaction
Tube A | Content | Volume |
Template | 10uL | |
10X Ligase Buffer | 1uL | |
7ng/uL CPG30K | 7.24uL | |
RNAse free H2O | 1.76uL | |
Mineral Oil | 20uL | |
Total Volume | 40uL |
Tube B | Content | Volume |
Template | 10uL | |
10X Ligase Buffer | 1uL | |
0.07ng/uL CPG30K | 4.29uL | |
RNAse free H2O | 4.71uL | |
Mineral Oil | 20uL | |
Total Volume | 40uL |
- 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 4 repeat 8 times 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