Matt:LabNotes/2016-11-15: Difference between revisions
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**Ampligase: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O | **Ampligase: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O | ||
**NTC & SplintR: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O | **NTC & SplintR: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O | ||
**T4: T4 DNA Ligase + 1ul 10X T4 Buffer + ATP + H2O | **T4: 1ul T4 DNA Ligase + 1ul 10X T4 Buffer + 5.5ul ATP + 2.5ul H2O | ||
***Thermo T4 DNA Ligase HC, 30 Weiss U/ul | |||
***ATP 10mM | |||
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===qPCR=== | ===qPCR=== |
Revision as of 22:00, 17 November 2016
In vitro capture with CA12kOct2016 V4 SplintR Ligase Test 2
- gDNA and UHRR
- SplintR and T4 DNA Ligase for HBRR
Reference
Experimental Outline
- CA12kOct2016 V4 Padlock Probe capture of gDNA and RNA
- Sequence to quantify probe efficiencies
Sample Groups
- RNA template = 100ng Universal Human Reference RNA (UHRR 740000-41)
- DNA template = 300ng gDNA 12878
- NTC - 9.9ng V4 - SplintR
- 150ng RNA - 9.9ng V4 - SplintR
- 150ng RNA - 9.9ng V4 - T4
- 160ng gDNA - 9.9ng V4 - Ampligase
Experiment
V4 Padlock Probe Capture
- Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc)
- DNA is 12878 80.3ng/ul
- V4 padlock probes: *20.7nM
Sample | RNA (50ng/ul) | DNA (80.3ng/ul) | V4 | 10X Buffer | H2O | Total |
NTC | 0 | 0 | 10 | 3 | 17 | 30 |
SplintR | 3 | 0 | 10 | 3 | 14 | 30 |
T4 | 3 | 0 | 10 | 3 | 14 | 30 |
Ampligase | 0 | 2 | 10 | 3 | 15 | 30 |
- Added 50ul mineral oil on top
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- -> add 3ul Enzyme mix
- For Ampligase: -> 55C 20h -> 94C 2min -> add 2ul Exo I/III -> 37C 2h -> 94C 2min -> 4C hold
- For SplintR and T4: -> 37C 15min -> 94C 2min -> add 2ul Exo I/III -> 37C 2h -> 94C 2min -> 4C hold
- Add 3ul Enzyme mix prepared on ice!
- Ampligase: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
- NTC & SplintR: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O
- T4: 1ul T4 DNA Ligase + 1ul 10X T4 Buffer + 5.5ul ATP + 2.5ul H2O
- Thermo T4 DNA Ligase HC, 30 Weiss U/ul
- ATP 10mM