Matt:LabNotes/2015-5-18

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Revision as of 02:47, 19 May 2015 by >Mzcai (→‎gDNA)
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in vitro Capture with CA12k_Nov2014 + suppressor oligo v2 Probe Set

Sample Groups

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

gDNA

Probe:target 1000:1 '
Probe size 3514 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 (3514, 150nt) 1.71585106x10^8 g/mol
Amount Probe req'd 26.4 ng
  • 461nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 22.5 ng/ul
    • Dilute to 16ul of 2.25ng/ul
    • 10X less probes in cDNA than gDNA
  • Make suppressor oligo pools with 5nM and 50nM of each supp oligos
    • 1000-fold more of each suppressor oligo: 1.5385x10^-16 mole * 1000 = 1.5385x10^-13 mole = 1.5385x10^-4 nmole = 50 nM * 3.1ul
    • 10X less probes in cDNA than gDNA
Sample # Sample Description Probes Target 1000X Supp Oligos 10X Ampligase Buffer H2O Total
1 gDNA 12 3.8 3.1 (50nM each) 3 8 30
2 cDNA dT 1.2 20 3.1 (5nM each) 3 2.7 30
3 cDNA N9 1.2 20 3.1 (5nM each) 3 2.7 30
4 NTC 1.2 20 3.1 (5nM each) 3 2.7 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-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.