Matt:LabNotes/2016-6-24

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Revision as of 23:56, 24 June 2016 by >Mzcai (Created page with "==in vitro Capture with TB12k_Apr2016 V7== *Will capture gDNA from mouse embryonic fibroblasts (got from Andrew) ===Sample Groups=== #gDNA MEF (77ng/ul) #Negative Control ==...")
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in vitro Capture with TB12k_Apr2016 V7

  • Will capture gDNA from mouse embryonic fibroblasts (got from Andrew)

Sample Groups

  1. gDNA MEF (77ng/ul)
  2. Negative Control

gDNA

Probe:target 1000:1 '
Probe size 1808 probes
DNA template 300 ng
gDNA MW 1.8x10^12 g/mol
gDNA (300ng) 1.66x10^-19 mol
Probe (1000:1) 1.66x10^-16 mol
Probe MW (1808, 160nt) 8.7997168x10^7 g/mol
Amount Probe req'd 14.6 ng


Sample # Sample Description Probes Target 10X Ampligase Buffer H2O Total
1 gDNA 3.9 3 30
2 NTC 0 3 30
  • Add 40ul Mineral Oil on top

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
    • In BioRad Thermalcycler program says: -0.2C per cycle every 30sec
  • -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
  • -> 55 C 20h (actually 24h)-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.