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]
- 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
- 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 | 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)