Matt:LabNotes/2016-6-24

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in vitro Capture with TB12k_Apr2016 V7[edit]

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

Sample Groups[edit]

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

gDNA[edit]

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 0.8 3.9 3 22.3 30
2 NTC 0.8 0 3 26.2 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.
    • Tiffany accidentally stopped reaction after 30min of Exo I/III digestion
    • She let it run 2min at 94C and then put it in fridge
    • Soon after, she called and I asked her to add 2ul of fresh Exo I/III and finish out the protocol with 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 1
10x AmpLigase Buffer 10 x 1 x 1
H2O 8
Total 10

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
Sample Index Forward Primer Reverse Primer
gDNA 1 ISB_CA_AF ISB_CA_AR.T1
NTC 2 ISB_CA_AF ISB_CA_AR.T2

PCR Test[edit]

Components 1X Volume 2.5X Volume
Captured template 1 0
10uM Forward Primer 0.4 1
10uM Reverse Primer 0.4 0
2X KAPA SYBG MM 12.5 31.25
H2O 10.7 26.75
Total 25 62.5
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21

File:2016-06-27 TB12kApr2016 V7 intubecapture testPCR.jpg

PCR[edit]

Components 1X Volume 2.5X Volume
Captured template 12 0
10uM Forward Primer 2 5
10uM Reverse Primer 2 0
2X KAPA SYBG MM 50 125
H2O 34 85
Total 100 250
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min -> 4C hold

File:2016-06-27 TB12kApr2016 V7 intubecapture PCR.jpg

  • Bead purification with 0.8:1 Beads to amplicon volume ratio
    • Eluted with 50ul total for each sample

PAGE Check[edit]

File:2016-06-28 TB12kApr16V7 CapturegDNA.jpg

  • Labeled tubes and put in "Non-quantified Sequencing Libraries"
    • Sample: MC20160628_TB12kApr16V7_gDNA-1 (MC160628_TB12kAprV7_gDNA-1)