Matt:LabNotes/2017-6-19: Difference between revisions
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'''Program'''<br> | '''Program'''<br> | ||
*95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h | *95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h | ||
**Should have added RiboLock after reached 55C since it would be denatured at 95C | |||
*Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer) | *Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer) | ||
*SplintR: add 3ul SplintR enzyme mix ( | *SplintR: add 3ul SplintR enzyme mix (10ul SplintR + 2ul 10X SplintR Buffer + 4ul Ribolock + 4ul H2O) | ||
*Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold | *Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold | ||
*SplintR: -> 37 C | *SplintR: -> 37 C 30min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold | ||
*Zymo columns | *Zymo columns | ||
**Elute 10ul | **Elute 10ul | ||
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====AmpLigase enzyme mix==== | ====AmpLigase enzyme mix==== |
Revision as of 18:56, 20 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
- Should have added RiboLock after reached 55C since it would be denatured at 95C
- Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- SplintR: add 3ul SplintR enzyme mix (10ul SplintR + 2ul 10X SplintR Buffer + 4ul Ribolock + 4ul H2O)
- Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold
- SplintR: -> 37 C 30min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold
- Zymo columns
- Elute 10ul