Matt:LabNotes/2015-5-18
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in vitro Capture with CA12k_Nov2014 + suppressor oligo v2 Probe Set[edit]
- Last time captured HBRR cDNA with 39 suppressors was not effective for most highly captured genes so made 5 more with new design that is hopefully more effective
- Will capture cDNA from BA8 tissue
- Using RNA from Blue's bulk RNA-Seq experiment
- 2 versions of cDNA:
- RT with dT (poly dT)
- RT with N9 (random nonamer)
Sample Groups[edit]
- gDNA 12878 (80.3ng/ul)
- Also a positive control and will be done using same volumes/amounts as all previous experiments
- cDNA dT BA8
- ~1/10th of gDNA amount
- cDNA N9 BA8
- ~1/10th of gDNA amount
- Negative Control
- From cDNA synthesis procedure where no RTase was added
- If see capture product, then there was DNA in Blue's RNA sample
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]
- Continued here: Matt:LabNotes/2015-7-12