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

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
No edit summary
>Alan6017518
Line 20: Line 20:
*47.1ng/ul *20ul = 942ng
*47.1ng/ul *20ul = 942ng
*Stored in Maytag -20C (Alan Misc. #9)
*Stored in Maytag -20C (Alan Misc. #9)
==BSPP capture set up==
==Probe calculations==
===illumina methylation seq pool 155===
===ppMALAT1===
===GP1_V6===
==Capture setup==
===illumina methylation seq pool 155 probes setup===
===illumina methylation seq pool 155 probes setup===
===ppMALAT1 setup===
===ppMALAT1 setup===
===GP1_V6 setup===
===GP1_V6 setup===

Revision as of 00:20, 16 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
  • Stored in Maytag -20C (Alan Misc. #9)

Probe calculations

illumina methylation seq pool 155

ppMALAT1

GP1_V6

Capture setup

illumina methylation seq pool 155 probes setup

ppMALAT1 setup

GP1_V6 setup