Matt:LabNotes/2015-1-21

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CA12k_Nov2014 V4 and V7 in vitro Capture[edit]

Calculate Probes Needed[edit]

V4[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

V7[edit]

Probe:target 1000:1 '
Probe size 2486 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 (2486, 150nt) 1.21388894x10^8 g/mol
Amount Probe req'd 18.7 ng

Probes, Target and Ampligase Buffer Mix[edit]

Sample # Sample Description Probes Target 10X Ampligase Buffer H2O Total
1 V4 - gDNA 2.5 3.8 3 20.7 30
2 V4 - cDNA 2.5 3.8 3 20.7 30
3 V4 - NTC 2.5 0 3 24.5 30
4 V7 - gDNA 1 15 3 11 30
5 V7 - cDNA 1 15 3 11 30
6 V7 - NTC 1 0 3 26 30

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
  • -> 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.
  • FORGOT TO ADD MINERAL OIL; added mineral oil after adding 3ul Ampligase mix

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
5 2 ISB_CA_AF ISB_CA_AR.T2
6 3 ISB_CA_AF ISB_CA_AR.T3

PCR Test[edit]

Components 1X Volume 6.5X Volume
Captured template 1 0
10uM Forward Primer 0.4 2.6
10uM Reverse Primer 0.4 0
2X KAPA SYBG MM 12.5 81.25
H2O 10.7 69.55
Total 25 153.4
  • Aliquot 23.6ul from 6.5X 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)x15 -> 72C 3min

File:012314 CA12kNov14 CaptureSequence test.JPG

  • Looks good; NTC are both negative so I won't amplify on next PCR

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)x13 -> 72C 3min

File:012614 CA12kNov14 CaptureSequencePCR.JPG

  • Bead purification with 1.5:1 Beads to amplicon volume ratio
    • Eluted with 50ul total for each sample

PAGE Quantification[edit]

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

File:2015-01-28 CA12k Nov2014 V4V7 SequenceQuant.jpg

  • Sample1 (V4 - gDNA):40ng/ul
  • Sample2 (V4 - cDNA):41ng/ul
  • Sample4 (V7 - gDNA):10ng/ul
  • Sample5 (V7 - cDNA):12ng/ul

concentration calculations