Matt:LabNotes/2017-6-19: Difference between revisions
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Revision as of 22:11, 19 June 2017
Agi15k_Feb2017 V4 + 5% formamide in vitro Capture
Calculate Probes Needed
V4
Probe:target | 1000:1 | ' |
Probe size | 4998 | 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 (4998, 157nt) | 2.387x10^8 | g/mol |
Amount Probe req'd | 36.7 | ng |
Probes, Target and Ampligase Buffer Mix
- gDNA: 12878 (80.3ng/ul)
- UHRR: 740000-41 (1ug/ul)
- Agi15k_Feb2017 Probe Design
- V4 Probe Production (10.3 ng/ul)
Sample | Condition | V4 (10.3ng/ul) | 12878 DNA (80.3ng/ul) | UHRR (1ug/ul) | Ampligase Buffer | SplintR Buffer | Formamide or DMF | Ribolock 40U/ul | H2O | Total |
1 | Ampligase DNA | 3.6 | 3.8 | 0 | 3 | 0 | 0 | 0 | 19.6 | 30 |
2 | Ampligase NTC | 3.6 | 0 | 0 | 3 | 0 | 0 | 0 | 23.4 | 30 |
3 | SplintR 10% DMF DNA | 3.6 | 3.8 | 0 | 0 | 3 | 3 | 0 | 16.6 | 30 |
4 | SplintR 10% DMF RNA | 3.6 | 0 | 1 | 0 | 3 | 3 | 0.5 | 18.9 | 30 |
5 | SplintR 10% DMF NTC | 3.6 | 0 | 0 | 0 | 3 | 3 | 0 | 20.4 | 30 |
6 | SplintR 5% formamide DNA | 3.6 | 3.8 | 0 | 0 | 3 | 1.5 | 0 | 18.1 | 30 |
7 | SplintR 5% formamide RNA | 3.6 | 0 | 1 | 0 | 3 | 1.5 | 0.5 | 20.4 | 30 |
8 | SplintR 5% formamide NTC | 3.6 | 0 | 0 | 0 | 3 | 1.5 | 0 | 21.9 | 30 |
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h