AlanFung:LabNotes/CTCF/2010-6-7: Difference between revisions

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>Alan6017518
Line 43: Line 43:
*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