AlanFung:LabNotes/Capturing/2014-1-14: Difference between revisions

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==Bisulfite Conversion of Jurkat gDNA==
==Bisulfite Conversion of Jurkat gDNA==
*Bisulfite conversion with zymo methylation gold  
*Bisulfite conversion with zymo methylation gold  
*Convert 500ng of Jurkat gDNA  
*Convert 500ng of Jurkat gDNA (2 rxn, 100ng total)
*Add 130ul of lighting conversion reagent to 5ul of jurkat gDNA and 15ul of ddH2O  
*Add 130ul of lighting conversion reagent to 5ul of jurkat gDNA and 15ul of ddH2O  
*Mix and centrifuge
*Mix and centrifuge

Revision as of 01:30, 15 January 2014

Capturing Bisulfite Converted Jurkat gDNA w GP1 V6 probe set

Capturing Jurkat gDNA w ppMALT1

Bisulfite Conversion of Jurkat gDNA

  • Bisulfite conversion with zymo methylation gold
  • Convert 500ng of Jurkat gDNA (2 rxn, 100ng total)
  • Add 130ul of lighting conversion reagent to 5ul of jurkat gDNA and 15ul of ddH2O
  • Mix and centrifuge
1. 98°C for 8 minutes 
2. 54°C for 60 minutes 
3. 4°C hold
  • Add 600ul M-binding buffer to column
  • load the sample into column, mix by inverting column and centrifuge at 14000rpm for 30sec
  • discard flow through
  • add 100ul m-wash buffer to the column and centrifuge at 14000rpm for 30sec
  • add 200ul L-Desulphonation buffer to the column and sit at RT for 20 minutes and centrifuge at 14000rpm for 30sec
  • add 200ul of m-wash buffer to the column and centrifuge at 14000rpm for 30sec
  • Add 10ul of EBuffer to column sit for 5 mintues and centrifuge at 14000rpm for 30sec (2 rxn total)

ssDNA Qubit assay

47.1ng/ul *20ul = 942ng