Matt:LabNotes/2016-11-2: Difference between revisions
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**120ul 3M NaOAc pH 5.2-5.5 | **120ul 3M NaOAc pH 5.2-5.5 | ||
*Vortexed and put in -80C for | *Vortexed and put in -80C for overnight | ||
*Centrifuged at 3000rpm at 4C for 30 min | *Centrifuged at 3000rpm at 4C for 30 min | ||
*Discarded supernatant and added 800ul of cold 80% EtOH | *Discarded supernatant and added 800ul of cold 80% EtOH | ||
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*Discarded supernatant and air-dried for 5 min in hood | *Discarded supernatant and air-dried for 5 min in hood | ||
*Resuspended DNA with 100ul H2O | *Resuspended DNA with 100ul H2O | ||
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===Qia Column Purification=== | ===Qia Column Purification=== | ||
*8 columns elute 50ul each | *8 columns elute 50ul each |
Revision as of 18:44, 7 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 File:20161102 CA12kOct2016V4 ExpansionPCR.JPG
- Purified with 2 Qiagen PCR columns (50ul PCR product each)
- Elute with 50ul H2O
- Quantify with Qubit dsDNA:
- 2.6ng/ul / (195bp*607.4Da/bp+157.9Da) = 21.9 nmol/L
- Dilute to 10nM
- 21.9nM x 99ul = 10nM x 216.8ul
- Add 117.8ul H2O
Production PCR
- V4 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Oct2016_V4 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V4U (100uM) | 0.4 | 20 |
AP2V4 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
- Did one plate - 96 wells 50ul each
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold File:20161103 CA12kOct2016V4 ProductionPCR.JPG
- Stopped after 14 cycles
- Based on touchscreen image curve probably peaked at cycle 13 and started to go down just a little at cycle 14
- Around cycle 12 the PCR curve started to look tilted like in the image here
- It started with about half the wells with no obvious pattern which wells
- Right at cycle 14 all the remaining normal curves also became tilted
EtOH Precipitation
- 8 5-ml tubes each with
- 1,200ul PCR product
- 3000ul 100% EtOH
- 4ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 3000rpm at 4C for 30 min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
- Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O