Matt:LabNotes/2016-11-2: Difference between revisions
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*50ul per PCR tube | *50ul per PCR tube | ||
'''Program''' | '''Program''' | ||
95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x | 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 23 -> 72C 2min -> 15C hold | ||
*Purified with 2 Qiagen PCR columns (50ul PCR product each) | *Purified with 2 Qiagen PCR columns (50ul PCR product each) | ||
**Elute with 50ul H2O | **Elute with 50ul H2O |
Revision as of 23:40, 2 November 2016
CA12k_Oct2016 Probe Preparation
- Received CA12k_Oct2016_Oligos2Order_FINAL in 80ul TE Buffer
- 47.35ng/ul (3.79ug total)
- Calculate MW of 195 nt probe = 195nt * 303.7D/nt + 79D = 59,300.5 g/mol
- 47.35 ng/ul * nmol/59300.5ng = 0.0007985 nmol/ul = 0.8 pmol/ul = 0.8 uM
- 47.35ng/ul (3.79ug total)
- Make 12.5ul aliquots in PCR strip tubes for future
- CA12k_Oct2016 Probe Design
- May also see this probe set called TB12k_Oct2016 in my notes
Probeset | Amplification | # Probes | # Barcodes | # Genes |
Human Brain | V4 | 4,978 | 391 | 368 |
Mouse Brain | V7 | 3,602 | 342 | 319 |
Mouse Embryo | V6 | 2,469 | 251 | 220 |
Lung Cancer | V8 | 105 | 105 | 85 |
Expansion PCR Test V4
- NTC
Component | Volume |
Seed oligo | 0 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 10.5 |
Total | 25 |
- Pos Control
Component | Volume |
TB12k_Apr2016 Seed oligo (100X Dilution) | 2.5 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 8 |
Total | 25 |
- 50nM CA12k_Oct2016
Component | Volume |
Seed oligo (800nM) | 1.5 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 9 |
Total | 25 |
- 100nM CA12k_Oct2016
Component | Volume |
Seed oligo (800nM) | 3.125 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 7.375 |
Total | 25 |
Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold File:20161102 CA12kOct2016V4 ExpansionPCRtest.JPG
Expansion PCR V4
Component | Volume |
Seed oligo (800nM) | 12.5 |
F/R V4 Primer Mix (10uM) | 8 |
2X Kapa MM | 50 |
H2O | 29.5 |
Total | 100 |
- 50ul per PCR tube
Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 23 -> 72C 2min -> 15C hold
- Purified with 2 Qiagen PCR columns (50ul PCR product each)
- Elute with 50ul H2O
- Quantify with Qubit dsDNA: