AlanFung:LabNotes/CTCF/2010-6-7: Difference between revisions
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*Elute with 50ul buffer | *Elute with 50ul buffer | ||
Perform A-tailing protocol | Perform A-tailing protocol (6/8/10) | ||
*Add in 6ul dA-tailing Reaction buffer | *Add in 6ul dA-tailing Reaction buffer | ||
*Add in 4ul klenow fragment | *Add in 4ul klenow fragment | ||
Line 51: | Line 51: | ||
Purification using qiaquick column | Purification using qiaquick column | ||
*Elute with 50ul buffer | *Elute with 50ul buffer | ||
Adapter ligation | |||
*Mix 1ul methylation adapter | |||
*Mix 5ul ligase | |||
*Mix 14ul ligase buffer | |||
*Incubate @20c for 30mins |
Revision as of 17:34, 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