AlanFung:LabNotes/Capturing/2014-1-14

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Revision as of 00:18, 16 January 2014 by >Alan6017518 (→‎ssDNA Qubit assay)
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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
  • Stored in Maytag -20C (Alan Misc. #9)

illumina methylation seq pool 155 probes setup

ppMALAT1 setup

GP1_V6 setup