Matt:LabNotes/2016-11-2: Difference between revisions
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===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
*Put cut out gel in | *Put cut out gel in 4 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT. | ||
*Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip. | *Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip. | ||
*Added 450 ul of 1X TE buffer to each | *Added 450 ul of 1X TE buffer to each | ||
Line 304: | Line 304: | ||
*Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min | *Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min | ||
*Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min | *Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min | ||
*Transferred spnt to fresh 1.5 mL tube | *Transferred spnt to fresh 1.5 mL tube | ||
*Precipitated in | *Precipitated in 4 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2 | ||
*Vortexed and placed the | *Vortexed and placed the 4 tubes at -80C for 30min | ||
*Spun | *Spun 4 tubes at 10,000rpm at 4C for 30min | ||
*Discard supernatant and add 750ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min | *Discard supernatant and add 750ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min | ||
*Discard supernatant and let dry in hood for 10min | *Discard supernatant and let dry in hood for 10min |
Revision as of 21:52, 8 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 50ul H2O (usually 100ul but because only did 5ml instead of 10ml production PCR this time)
Qia Column Purification
- 4 columns elute 50ul each
- Nanodrop: 208.8 ng/uL x 200uL = 41.76ug
Lambda Exo
- Divide into 4 pcr tubes of 100ul (ideally should be <10ug each)
Components | Volume |
Amplicon | 50 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 30 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with 4 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 96.8ng/ul x 160ul = 15.49ug (74% yield)
Remove Amplification Adapters
- Save 4ul for gel check of size
USER
- Split into 3 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 52 |
USER | 5 |
10X DpnII Buffer | 8 |
H2O | 15 |
Total | 80 |
- Incubate at 37C for 2.5 hours
DpnII
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 2 |
100uM RE-DpnII_V4 guide oligo | 5 |
H2O | 8 |
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight (~15hrs)
Zymo Column Purification
- Eluted 20ul each column (60ul total)
- Nanodrop
- 87.3 ng/ul x 60ul = 5.2ug (34% yield lower than normal)
Gel Check
- Pre-run 12 well TBU gel for 20min at 250V
- Rinse wells
- Ladder
- 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X TBU Loading Dye
- User/DpnII-Digested
- 2ul Sample + 2ul 2X TBU Loading Dye
- Lambda-Digested
- 2ul Sample + 2ul 2X TBU Loading Dye
- Pre-heat samples to 75C for 10min and then put on ice
- Load samples in middle lanes of gel
- Run for 33min at 210V
File:2016-11-08 CA12kOct2016 V4 GelCheck.jpg
PAGE Size Selection
- Run 2 gels
- 200V for 40min
Components | 2X Volume |
V4 Probes | 60 |
TBE-Urea Buffer 2X | 60 |
Components | 2X Volume |
Low Mass Ladder | 2 |
TBE-Urea Buffer 2X | 10 |
H2O | 8 |
Total | 20 |
[[File:|350px]]
[[File:|350px]]
EtOH Precipitation
- Put cut out gel in 4 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT.
- Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
- Added 450 ul of 1X TE buffer to each
- Vortexed for 60min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min
- Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transferred spnt to fresh 1.5 mL tube
- Precipitated in 4 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
- Vortexed and placed the 4 tubes at -80C for 30min
- Spun 4 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 750ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
- Discard supernatant and let dry in hood for 10min
- Resuspend each tube with 10ul and combine
Qubit ssDNA
20.2ng/ul x 74ul = 1,494.8ng 20.2ng/ul / (157nt x 303.7Da/nt + 79Da) = 423nM -->