Matt:LabNotes/2016-6-24
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in vitro Capture with TB12k_Apr2016 V7
- Will capture gDNA from mouse embryonic fibroblasts (got from Andrew)
Sample Groups
- gDNA MEF (77ng/ul)
- Negative Control
gDNA
Probe:target | 1000:1 | ' |
Probe size | 1808 | probes |
DNA template | 300 | ng |
gDNA MW | 1.8x10^12 | g/mol |
gDNA (300ng) | 1.66x10^-19 | mol |
Probe (1000:1) | 1.66x10^-16 | mol |
Probe MW (1808, 160nt) | 8.7997168x10^7 | g/mol |
Amount Probe req'd | 14.6 | ng |
Sample # | Sample Description | Probes | Target | 10X Ampligase Buffer | H2O | Total |
1 | gDNA | 3.9 | 3 | 30 | ||
2 | NTC | 0 | 3 | 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.