AlanFung:LabNotes/CTCF/2010-6-7: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
Line 57: | Line 57: | ||
*Mix 14ul ligase buffer | *Mix 14ul ligase buffer | ||
*Incubate @20c for 30mins | *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 |
Revision as of 18:41, 8 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