Matt:LabNotes/2015-5-18: Difference between revisions
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>Mzcai m (→gDNA) |
>Mzcai mNo edit summary |
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===Add Sequence Adapters PCR=== | ===Add Sequence Adapters PCR=== | ||
====Primers==== | ====Primers==== | ||
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|- | |- | ||
| 4||1||ISB_CA_AF||ISB_CA_AR.T1 | | 4||1||ISB_CA_AF||ISB_CA_AR.T1 | ||
|} | |} | ||
====PCR Test==== | ====PCR Test==== | ||
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| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''1X Volume''' | | align="center" style="background:#f0f0f0;"|'''1X Volume''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''4.5X Volume''' | ||
|- | |- | ||
| Captured template||1||0 | | Captured template||1||0 | ||
|- | |- | ||
| 10uM Forward Primer||0.4|| | | 10uM Forward Primer||0.4||1.8 | ||
|- | |- | ||
| 10uM Reverse Primer||0.4||0 | | 10uM Reverse Primer||0.4||0 | ||
|- | |- | ||
| 2X KAPA SYBG MM||12.5|| | | 2X KAPA SYBG MM||12.5||56.25 | ||
|- | |- | ||
| H2O||10.7|| | | H2O||10.7||48.15 | ||
|- | |- | ||
| Total||25|| | | Total||25||106.2 | ||
|} | |} | ||
*Aliquot 23.6ul from | *Aliquot 23.6ul from 4.5X master mix and add 1ul captured template and 0.4ul corresponding reverse primer | ||
Program | Program | ||
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min | 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min | ||
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[[ | [[ | 650px]] | ||
*Looks good; NTC are both negative so I won't amplify on next PCR | *Looks good; NTC are both negative so I won't amplify on next PCR | ||
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| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''1X Volume''' | | align="center" style="background:#f0f0f0;"|'''1X Volume''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''3.5X Volume''' | ||
|- | |- | ||
| Captured template||12||0 | | Captured template||12||0 |
Revision as of 19:42, 21 May 2015
in vitro Capture with CA12k_Nov2014 + suppressor oligo v2 Probe Set
- Last time captured HBRR cDNA with 39 suppressors was not effective for most highly captured genes so made 5 more with new design that is hopefully more effective
- Will capture cDNA from BA8 tissue
- Using RNA from Blue's bulk RNA-Seq experiment
- 2 versions of cDNA:
- RT with dT (poly dT)
- RT with N9 (random nonamer)
Sample Groups
- gDNA 12878 (80.3ng/ul)
- Also a positive control and will be done using same volumes/amounts as all previous experiments
- cDNA dT BA8
- ~1/10th of gDNA amount
- cDNA N9 BA8
- ~1/10th of gDNA amount
- Negative Control
- From cDNA synthesis procedure where no RTase was added
- If see capture product, then there was DNA in Blue's RNA sample
gDNA
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 |
- 461nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 22.5 ng/ul
- Dilute to 16ul of 2.25ng/ul
- 10X less probes in cDNA than gDNA
- Make suppressor oligo pools with 5nM and 50nM of each supp oligos
- 1000-fold more of each suppressor oligo: 1.5385x10^-16 mole * 1000 = 1.5385x10^-13 mole = 1.5385x10^-4 nmole = 50 nM * 3.1ul
- 10X less probes in cDNA than gDNA
Sample # | Sample Description | Probes | Target | 1000X Supp Oligos | 10X Ampligase Buffer | H2O | Total |
1 | gDNA | 12 | 3.8 | 3.1 (50nM each) | 3 | 8 | 30 |
2 | cDNA dT | 1.2 | 20 | 3.1 (5nM each) | 3 | 2.7 | 30 |
3 | cDNA N9 | 1.2 | 20 | 3.1 (5nM each) | 3 | 2.7 | 30 |
4 | NTC | 1.2 | 20 | 3.1 (5nM each) | 3 | 2.7 | 30 |
- Add 40ul Mineral Oil on top
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- In BioRad Thermalcycler program says: -0.2C per cycle every 30sec
- -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- -> 55 C 20h (actually 24h)-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
AmpLigase enzyme mix
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
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 |
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 |
PCR Test
Components | 1X Volume | 4.5X Volume |
Captured template | 1 | 0 |
10uM Forward Primer | 0.4 | 1.8 |
10uM Reverse Primer | 0.4 | 0 |
2X KAPA SYBG MM | 12.5 | 56.25 |
H2O | 10.7 | 48.15 |
Total | 25 | 106.2 |
- Aliquot 23.6ul from 4.5X 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