Matt:LabNotes/2016-6-5

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in vitro Capture with TB12k_Apr2016 V4[edit]

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

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
    • cDNA N9 BA8
    • ~1/10th of gDNA amount
  2. Negative Control

gDNA[edit]

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.

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
gDNA 1 ISB_CA_AF ISB_CA_AR.T1
cDNA 2 ISB_CA_AF ISB_CA_AR.T2
NTC 3 ISB_CA_AF ISB_CA_AR.T3

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
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21

File:TB12k Apr2016 V4 Capture PCR test.JPG

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
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x26 -> 72C 3min -> 4C hold
  • Take gDNA out after 25 cycles
  • Take cDNA out after 30 cycles

File:TB12k Apr2016 V4 Capture PCR.JPG

  • 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:2016-06-13 TB12kApr2016 V4 intubecapture gelcheck.jpg

  • Bead purification did not work try again with 0.8:1 bead:sample ratio

File:2016-06-13 TB12kApr2016 V4 intubecapture gelcheckRnd2.jpg

  • Labeled tubes and put in "Non-quantified Sequencing Libraries"
    • Sample1: MC20160610_TB12kApr16_gDNA-1 (MC160610_TB12kApr_gDNA-1)
    • Sample2: MC20150610_TB12kApr16_cDNA-2 (MC160610_TB12kApr_cDNA-2)