Matt:LabNotes/2017-5-4
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Agi15k_Feb2017 V4 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)
- 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 | H2O | Total |
1 | Ampligase DNA | 3.6 | 3.8 | 0 | 3 | 0 | 0 | 19.6 | 30 |
2 | Ampligase NTC | 3.6 | 0 | 0 | 3 | 0 | 0 | 23.4 | 30 |
3 | SplintR DNA | 3.6 | 3.8 | 0 | 0 | 3 | 0 | 19.6 | 30 |
4 | SplintR RNA | 3.6 | 0 | 1 | 0 | 3 | 0 | 22.4 | 30 |
5 | SplintR NTC | 3.6 | 0 | 0 | 0 | 3 | 0 | 23.4 | 30 |
6 | SplintR 20% formamide DNA | 3.6 | 3.8 | 0 | 0 | 3 | 6 | 13.6 | 30 |
7 | SplintR 20% formamide RNA | 3.6 | 0 | 1 | 0 | 3 | 6 | 16.4 | 30 |
8 | SplintR 20% formamide NTC | 3.6 | 0 | 0 | 0 | 3 | 6 | 17.4 | 30 |
Program
- gDNA: 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- UHRR RNA: 55C 20h
- gDNA: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- UHRR: add 3ul SplintR enzyme mix (13.5ul SplintR + 2.25ul 10X SplintR Buffer + 6.75ul H2O)
- gDNA: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold
- UHRR: -> 37 C 15min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold
- Zymo columns
AmpLigase enzyme mix
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume 10ul |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 1.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 1.00 |
H2O | 8.00 | ||||
Total | 10.00 |