AlanFung:LabNotes/CTCF/2010-6-7: Difference between revisions
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*Add 50ul of M-elution buffer directly to the column matrix (Volume can be adjusted, depending on the requirements) | *Add 50ul of M-elution buffer directly to the column matrix (Volume can be adjusted, depending on the requirements) | ||
*Centrifuge for 30sec to elute the DNA | *Centrifuge for 30sec to elute the DNA | ||
==Perform PCR to bisulfite converted DNA using Phusion/iProof 6/9/10== | |||
*Primer | |||
{| {{table}} | |||
|- | |||
| PCR_F||Sol. Amp. (Forward)||AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T||3'-Phosphorothioate bond||[2] | |||
|- | |||
| PCR_R||Sol. Amp. (Reward)||CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC*T||3'-Phosphorothioate bond||[2] | |||
|- | |||
|} | |||
{| {{table}} | |||
| ||1rxn||X2.2 | |||
|- | |||
| Phusion||50||110 | |||
|- | |||
| PCRF||0.5||1.1 | |||
|- | |||
| PCRR||0.5||1.1 | |||
|- | |||
| SYBG||0.8||1.76 | |||
|- | |||
| DNA||50|| | |||
|- | |||
| | |||
|} | |||
a.Set up the reaction system with Phusion High-Fidelity PCR master mix: | |||
2x Phusion master mix: 50ul | |||
100uM PCR_F 0.5ul | |||
100uM PCR_R 0.5ul | |||
50X SYBG I 0.8ul | |||
Captured DNA 50ul | |||
b.Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min. | |||
Terminate the reaction when the amplification curves approach to the plateau. |
Revision as of 16:17, 9 June 2010
Prepare PCR Product from Jurkat gDNA
ul | |
primer | 1 |
h2o | 7 |
template | 2 |
Econo 2x | 10 |
- Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C 60 sec) x 2 -> 72C 10min -> 15C hold
- Qiaquick Purification
- Nanodrop
P7-6.4ng/ul P10-5.4ng/ul
- Gel Quant
File:ZhangLab 2 2010-06-07 14hr 34min.jpg
Protocol
Obtain 100ng Jurkat DNA
- 100ng/(6.4ng/ul)=15.63ul
- 100ng/(5.4ng/ul)=18.52ul
P7 | P10 | |
Jurkat DNA PCR Product | 15.63 | 18.52 |
ddH2O | 69.37 | 66.48 |
End Repair Reaction Buffer (10x) | 10 | 10 |
Repair Enzyme Mix | 5 | 5 |
- Incubate 30 mins at 20C
Purification using qiaquick column
- Elute with 50ul buffer
Perform A-tailing protocol (6/8/10)
- Add in 6ul dA-tailing Reaction buffer
- Add in 4ul klenow fragment
- Incubate at 37C for 30mins
Purification using qiaquick column
- Elute with 50ul buffer
Adapter ligation
- Mix 1ul methylation adapter
- Mix 5ul ligase
- Mix 14ul ligase buffer
- Incubate @20c for 30mins
Purification using qiaquick column
- Elute with 50ul buffer
CT Conversion (6/3/10)
- Take 46ul to perfrom CT conversion
an additional 1m Bisulfite conversion of DNA
- Add 46ul of sample to 130ul of CT conversion reagent solution in a pcr tube
- Vortex the sample to mix
- Pulse centrifuge
- Perform
*98C for 8m *64C for 3.5hr *4C hold
- Add 600ul of M binding buffer into a column assembly
- Load sample(s) to the column
- Close the cap and mix by inverting the column several times
- Centrifuge at >10,000g for 30sec
- Discard the flow through
- Add 100ul of M-Wash Buffer to the column
- Centrifuge at full speed for 30sec
- Add 200ul of M-Desulphonation buffer to the column and let stand at RT for 20m
- Centrifuge at full speed for 30sec
- Add 100ul of M-Wash Buffer to the column
- Centrifuge at full speed for 30sec
- Place the column into a 1.5ml tube
- Add 50ul of M-elution buffer directly to the column matrix (Volume can be adjusted, depending on the requirements)
- Centrifuge for 30sec to elute the DNA
Perform PCR to bisulfite converted DNA using Phusion/iProof 6/9/10
- Primer
PCR_F | Sol. Amp. (Forward) | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T | 3'-Phosphorothioate bond | [2] |
PCR_R | Sol. Amp. (Reward) | CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC*T | 3'-Phosphorothioate bond | [2] |
1rxn | X2.2 | |
Phusion | 50 | 110 |
PCRF | 0.5 | 1.1 |
PCRR | 0.5 | 1.1 |
SYBG | 0.8 | 1.76 |
DNA | 50 | |
a.Set up the reaction system with Phusion High-Fidelity PCR master mix: 2x Phusion master mix: 50ul 100uM PCR_F 0.5ul 100uM PCR_R 0.5ul 50X SYBG I 0.8ul Captured DNA 50ul b.Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min. Terminate the reaction when the amplification curves approach to the plateau.