Matt:LabNotes/2015-4-28

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in vitro Capture with CA12k_Nov2014 Probe Set + Supp Oligos[edit]

HBRR cDNA Synthesis[edit]

  • Followed NEB E6300S protocol: [1]
  • Human Brain Reference RNA: [2]
    • Labeled concentration = 1ug/ul
  • 2 tubes for RNA sample and 1 tube with no RNA (replace with H2O) as negative control
Components Volume
RNA 0.5 ul (0.5 ug)
d(T)23VN (50 µM) 2 ul
H2O 5.5 ul
Total 8 ul
  • Denatured RNA for 5 min at 70C and then put on ice
  • Added the following to tubes
Components Volume
M-MuLV Reaction Mix 10 ul
M-MuLV Enzyme Mix 2 ul
Total 20 ul
  • Incubated for one hour at 42C
  • Inactivated enzyme for 5 min at 80C
  • Purified cDNA in one Qiagen PCR clean up column and NTC in another
    • Eluted 30ul each
  • Nanodrop w/ dsDNA setting:
    • cDNA: 30.7 ng/ul
    • Neg Ctrl: 10.2 ng/ul

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]

  • gDNA: 12878 (80.3ng/ul)
  • First strand cDNA from HBRR
  • CA12k_Nov2014 Probes
    • V4 819nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 40.0 ng/ul
    • V7 768nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 37.5 ng/ul
      • Diluted 1:2 with H2O for easier pipetting (final conc 20.0ng/ul and 18.75ng/ul)
  • NTC is the NTC from first strand cDNA synthesis
  • 5nM of each supp oligos (5 x 10^-6 nmol/ul)
      • 100-fold more of each suppressor oligo = 1.5385 x 10^-14 mol = 15 x 10^-6 nmol
Sample # Sample Description Probes Target 5nM Supp Oligos 10X Ampligase Buffer H2O Total
1 V4 - gDNA 1.3 3.8 3 3 18.9 30
2 V4 - cDNA 1.3 15 3 3 7.7 30
3 V4 - NTC 1.3 15 3 3 7.7 30
4 V7 - gDNA 1 3.8 3 3 19.2 30
5 V7 - cDNA 1 15 3 3 8 30
6 V7 - NTC 1 15 3 3 8 30
  • Sample 4, accidentally added an extra 9ul H2O so added 4ul 10X Ampligase Buffer
    • Added 4ul of AmpLigase enzyme mix as well (vs 3ul for others)
  • Add 40ul mineral oil on top

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.

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:20150501 CA12kNov2014 invitroPCRtest.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:20150502 CA12kNov2014 invitroPCR.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:2015-05-02 CA12kNov2014 invitro gelcheck.jpg

  • Labeled tubes and put in "Non-quantified Sequencing Libraries"
    • Sample1: MC20150502_CA12kNov14supp_V4gDNA-1
    • Sample2: MC20150502_CA12kNov14supp_V4cDNA-2
    • Sample3: MC20150502_CA12kNov14supp_V7gDNA-4
    • Sample4: MC20150502_CA12kNov14supp_V7cDNA-5