Matt:LabNotes/2015-4-28
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in vitro Capture with CA12k_Nov2014 Probe Set + Supp Oligos[edit]
HBRR cDNA Synthesis[edit]
- Followed NEB E6300S protocol: [1]
- Human Brain Reference RNA: [2]
- Labeled concentration = 1ug/ul
- 2 tubes for RNA sample and 1 tube with no RNA (replace with H2O) as negative control
Components | Volume |
RNA | 0.5 ul (0.5 ug) |
d(T)23VN (50 µM) | 2 ul |
H2O | 5.5 ul |
Total | 8 ul |
- Denatured RNA for 5 min at 70C and then put on ice
- Added the following to tubes
Components | Volume |
M-MuLV Reaction Mix | 10 ul |
M-MuLV Enzyme Mix | 2 ul |
Total | 20 ul |
- Incubated for one hour at 42C
- Inactivated enzyme for 5 min at 80C
- Purified cDNA in one Qiagen PCR clean up column and NTC in another
- Eluted 30ul each
- Nanodrop w/ dsDNA setting:
- cDNA: 30.7 ng/ul
- Neg Ctrl: 10.2 ng/ul
Calculate Probes Needed[edit]
V4[edit]
Probe:target | 1000:1 | ' |
Probe size | 3514 | 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 (3514, 150nt) | 1.71585106x10^8 | g/mol |
Amount Probe req'd | 26.4 | ng |
V7[edit]
Probe:target | 1000:1 | ' |
Probe size | 2486 | 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 (2486, 150nt) | 1.21388894x10^8 | g/mol |
Amount Probe req'd | 18.7 | ng |
Probes, Target and Ampligase Buffer Mix[edit]
- gDNA: 12878 (80.3ng/ul)
- First strand cDNA from HBRR
- CA12k_Nov2014 Probes
- V4 819nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 40.0 ng/ul
- V7 768nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 37.5 ng/ul
- Diluted 1:2 with H2O for easier pipetting (final conc 20.0ng/ul and 18.75ng/ul)
- NTC is the NTC from first strand cDNA synthesis
- 5nM of each supp oligos (5 x 10^-6 nmol/ul)
- 100-fold more of each suppressor oligo = 1.5385 x 10^-14 mol = 15 x 10^-6 nmol
Sample # | Sample Description | Probes | Target | 5nM Supp Oligos | 10X Ampligase Buffer | H2O | Total |
1 | V4 - gDNA | 1.3 | 3.8 | 3 | 3 | 18.9 | 30 |
2 | V4 - cDNA | 1.3 | 15 | 3 | 3 | 7.7 | 30 |
3 | V4 - NTC | 1.3 | 15 | 3 | 3 | 7.7 | 30 |
4 | V7 - gDNA | 1 | 3.8 | 3 | 3 | 19.2 | 30 |
5 | V7 - cDNA | 1 | 15 | 3 | 3 | 8 | 30 |
6 | V7 - NTC | 1 | 15 | 3 | 3 | 8 | 30 |
- Sample 4, accidentally added an extra 9ul H2O so added 4ul 10X Ampligase Buffer
- Added 4ul of AmpLigase enzyme mix as well (vs 3ul for others)
- Add 40ul mineral oil on top
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
AmpLigase enzyme mix[edit]
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume 30ul |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 2.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 2.00 |
H2O | 16.00 | ||||
Total | 20.00 |
Add Sequence Adapters PCR[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 |
Sample | Index | Forward Primer | Reverse Primer |
1 | 1 | ISB_CA_AF | ISB_CA_AR.T1 |
2 | 2 | ISB_CA_AF | ISB_CA_AR.T2 |
3 | 3 | ISB_CA_AF | ISB_CA_AR.T3 |
4 | 1 | ISB_CA_AF | ISB_CA_AR.T1 |
5 | 2 | ISB_CA_AF | ISB_CA_AR.T2 |
6 | 3 | ISB_CA_AF | ISB_CA_AR.T3 |
PCR Test[edit]
Components | 1X Volume | 6.5X Volume |
Captured template | 1 | 0 |
10uM Forward Primer | 0.4 | 2.6 |
10uM Reverse Primer | 0.4 | 0 |
2X KAPA SYBG MM | 12.5 | 81.25 |
H2O | 10.7 | 69.55 |
Total | 25 | 153.4 |
- Aliquot 23.6ul from 6.5X master mix and add 1ul captured template and 0.4ul corresponding reverse primer
Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
File:20150501 CA12kNov2014 invitroPCRtest.JPG
- Looks good; NTC are both negative so I won't amplify on next PCR
PCR[edit]
Components | 1X Volume | 4.5X Volume |
Captured template | 12 | 0 |
10uM Forward Primer | 2 | 9 |
10uM Reverse Primer | 2 | 0 |
2X KAPA SYBG MM | 50 | 225 |
H2O | 34 | 153 |
Total | 100 | 387 |
- Aliquot 86ul from 4.5X 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)x13 -> 72C 3min
File:20150502 CA12kNov2014 invitroPCR.JPG
- Bead purification with 1.5:1 Beads to amplicon volume ratio
- Eluted with 50ul total for each sample
PAGE Check[edit]
- Load 2ul of each sample + 2ul loading dye
File:2015-05-02 CA12kNov2014 invitro gelcheck.jpg
- Labeled tubes and put in "Non-quantified Sequencing Libraries"
- Sample1: MC20150502_CA12kNov14supp_V4gDNA-1
- Sample2: MC20150502_CA12kNov14supp_V4cDNA-2
- Sample3: MC20150502_CA12kNov14supp_V7gDNA-4
- Sample4: MC20150502_CA12kNov14supp_V7cDNA-5