Matt:LabNotes/2016-6-5
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in vitro Capture with TB12k_Apr2016 V4
- Will capture cDNA from BA8 tissue
- Using RNA from Blue's bulk RNA-Seq experiment
Sample Groups
- 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
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.