Matt:LabNotes/2016-11-2

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Revision as of 21:52, 8 November 2016 by >Mzcai (→‎EtOH Precipitation)
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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
  • Make 12.5ul aliquots in PCR strip tubes for future
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
  1. Ladder
    • 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X TBU Loading Dye
  2. User/DpnII-Digested
    • 2ul Sample + 2ul 2X TBU Loading Dye
  3. 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 -->