Revision as of 22:11, 18 November 2016 by >Mzcai
In vitro capture with CA12kOct2016 V4
- 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
qPCR
Primers
Primer
|
Sequence
|
Index #
|
ISB_CA_AF |
AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG |
|
ISB_CA_AR.T1 |
CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG |
Indx1
|
ISB_CA_AR.T2 |
CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG |
Indx2
|
ISB_CA_AR.T3 |
CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG |
Indx3
|
PCR Test for Non-Enzyme Digested Samples
Components
|
1X Volume
|
4X Volume
|
Captured template |
1 |
0
|
10uM ISB_CA_AF |
0.4 |
1.6
|
10uM ISB_CA_AR.T1 |
0.4 |
1.6
|
2X KAPA SYBG MM |
12.5 |
50
|
H2O |
10.7 |
42.8
|
Total |
25 |
96
|
- Aliquot 24ul from 4X master mix and add 1ul captured template
Program
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min