Matt:LabNotes/2016-6-5

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Revision as of 19:03, 5 June 2016 by >Mzcai (Created page with "==in vitro Capture with TB12k_Apr2016 V4== *Will capture cDNA from BA8 tissue **Using RNA from Blue's bulk RNA-Seq experiment ===Sample Groups=== #...")
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in vitro Capture with TB12k_Apr2016 V4

  • Will capture cDNA from BA8 tissue
    • Using RNA from Blue's bulk RNA-Seq experiment

Sample Groups

  1. gDNA 12878 (80.3ng/ul)
    • Also a positive control and will be done using same volumes/amounts as all previous experiments
    • cDNA N9 BA8
    • ~1/10th of gDNA amount
  2. Negative Control

gDNA

Probe:target 1000:1 '
Probe size 4978 probes
DNA templet 300 ng
gDNA MW 1.95x10^12 g/mol
gDNA (300ng) 1.5385x10^-19 mol
Probe (1000:1) 1.5385x10^-16 mol
Probe MW (4978, 157nt) 2.37748782x10^8 g/mol
Amount Probe req'd 36.6 ng
  • 423nM
    • Dilute 22ul to 3.66ng/ul (2.2ul probe + 19.8ul H2O)
    • 10X less probes in cDNA than gDNA
Sample # Sample Description Probes Target 1000X Supp Oligos 10X Ampligase Buffer H2O Total
1 gDNA 18 3.8 3 5.2 30
2 cDNA 1.8 20 3 5.2 30
3 NTC 1.8 20 3 5.2 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.