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

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==Prepare Exo Mix==
==Prepare Exo Mix==
*Mix 1:1 Exo I & Exo III (9ul total)
==PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 Ind1-4)==

Revision as of 22:58, 17 January 2014

Capturing Bisulfite Converted Jurkat gDNA w GP1 V6 probe set

  • probes prep notes LabNotes/Probe/2014-1-7

[[1]]

Capturing Jurkat gDNA w ppMALT1 ordered form IDT

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

GP1_V6            
Sample Conc. (ng/ul) Volume for 200ng (ul) H2O GP1_V6 10X ampligase Buffer Total
CT Jurkat 47.1 4.25 11.47 2.28 2 20
             
illumina methylation seq pool            
Sample Conc. (ng/ul) Volume for 200ng (ul) H2O illumina methylation seq pool 10X ampligase Buffer Total
CT Jurkat 47.1 4.25 10.38 3.37 2.00 20
         
ppMALAT1            
Sample Conc. (ng/ul) Volume for 200ng (ul) H2O ppMALAT1 10X ampligase Buffer Total
Jurkat 100 2 15.34 0.66 2.00 20
Negative Control n/a n/a 17.34 0.66 2.00 20

illumina methylation seq pool 155 probes setup

Program stored under Alan/aCPG
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 58C 20h 
-> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

GP1_V6 & ppMALAT1 setup

Program stored under Kun/CPG
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

Prepare SLN Mix

SLN Mix          
           
Components Stock Concentration Unit Final Concentration Unit Prepare Volume (ul)
AmpliTaq Stoffel 10 U/ul 2 U/ul 2.6
Ampligase 5 U/ul 0.5 U/ul 1.3
dNTP 1 mM 100 uM 1.3
10X Ampligase Buffer 10 X 1 X 1.3
H2O n/a n/a n/a n/a 6.5
Total n/a n/a n/a n/a 13

Prepare Exo Mix

  • Mix 1:1 Exo I & Exo III (9ul total)

PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 Ind1-4)