Matt:LabNotes/2016-11-15
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In vitro capture with CA12kOct2016 V4[edit]
- gDNA and UHRR
- SplintR and T4 DNA Ligase for HBRR
Reference[edit]
Experimental Outline[edit]
- CA12kOct2016 V4 Padlock Probe capture of gDNA and RNA
- Sequence to quantify probe efficiencies
Sample Groups[edit]
- 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[edit]
V4 Padlock Probe Capture[edit]
- 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[edit]
Primers[edit]
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[edit]
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
File:20161119 CA12kOct2016V4 invitroCapture PCRtest.JPG
PCR[edit]
Components | 1X Volume | 3.5X Volume |
Captured template | 12 | 0 |
10uM Forward Primer | 2 | 7 |
10uM Reverse Primer | 2 | 0 |
2X KAPA SYBG MM | 50 | 175 |
H2O | 34 | 119 |
Total | 100 | 301 |
- Indx1:T4
- Indx2:SplintR
- Indx3:Ampligase
- Aliquot 86ul from 3.5X master mix and add 12ul captured template and 2ul corresponding reverse primer
Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
File:20161203 invitro CA12kOct2016V4 PCR.png
AMPure Bead Purification[edit]
- 1:1 beads to amplicon volume ratio
- Eluted with 50ul total each sample
PAGE Check[edit]
- Load 2ul sample + 2ul loading dye
File:2016-12-15 CA12kOct2016 V4 invitroCapture Library.jpg
Sequencing Library[edit]
MC20161215_CA12kOct20161215_V4_T4_Indx1
MC20161215_CA12kOct20161215_V4_SplintR_Indx2
MC20161215_CA12kOct20161215_V4_Amp_Indx3