Matt:LabNotes/2015-5-18

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in vitro Capture with CA12k_Nov2014 + suppressor oligo v2 Probe Set[edit]

Sample Groups[edit]

  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[edit]

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 (actually 24h)-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

AmpLigase enzyme mix[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume 30ul
AmpLigase 5 U/ul 0.5 U/ul 2.00
10x AmpLigase Buffer 10 x 1 x 2.00
H2O 16.00
Total 20.00


Add Sequence Adapters PCR[edit]

Primers[edit]

Primer Sequence Index #
ISB_CA_AF AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG
ISB_CA_AR.T1 CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG Indx1
ISB_CA_AR.T2 CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG Indx2
ISB_CA_AR.T3 CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG Indx3
Sample Index Forward Primer Reverse Primer
1 1 ISB_CA_AF ISB_CA_AR.T1
2 2 ISB_CA_AF ISB_CA_AR.T2
3 3 ISB_CA_AF ISB_CA_AR.T3
4 1 ISB_CA_AF ISB_CA_AR.T1

PCR Test[edit]

Components 1X Volume 4.5X Volume
Captured template 1 0
10uM Forward Primer 0.4 1.8
10uM Reverse Primer 0.4 0
2X KAPA SYBG MM 12.5 56.25
H2O 10.7 48.15
Total 25 106.2
  • Aliquot 23.6ul from 4.5X master mix and add 1ul captured template and 0.4ul corresponding reverse primer
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21

File:20150521 CA12kNov14suppv2 invitroPCRtest.JPG

  • cDNA captured samples look barely better than NTC

PCR[edit]

Components 1X Volume 4.5X Volume
Captured template 12 0
10uM Forward Primer 2 9
10uM Reverse Primer 2 0
2X KAPA SYBG MM 50 225
H2O 34 153
Total 100 387
  • Aliquot 86ul from 4.5X master mix and add 12ul captured template and 2ul corresponding reverse primer
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x27 -> 72C 3min
  • Take gDNA out after 21 cycles
  • Take cDNA_dT out after 31 cycles
  • Take cDNA_N9 out after 27
  • NegCtrl had 34 cycles

File:20150521 CA12kNov14suppv2 invitroPCR.JPG

  • During PCR the curves had not decreased (only slightly plateau'd) when I took them out
  • Bead purification with 1.5:1 Beads to amplicon volume ratio
    • Eluted with 50ul total for each sample

PAGE Check[edit]

  • Load 2ul of each sample + 2ul loading dye

File:2015-05-22 CA12kNov2014suppv2 invitro gelcheck.jpg

  • Labeled tubes and put in "Non-quantified Sequencing Libraries"
    • Sample1: MC20150522_CA12kNov14suppv2_gDNA-1
    • Sample2: MC20150522_CA12kNov14suppv2_cDNAdT-2
    • Sample3: MC20150522_CA12kNov14suppv2_cDNARan-3
    • Sample4: MC20150522_CA12kNov14suppv2_NegCtrl-1

Analysis[edit]