Matt:LabNotes/2016-5-25

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TB12k_Apr2016 Probe Preparation[edit]

  • Probe Design
  • Received 195-mer oligos 5/25/26
    • Name: TB12k_Apr2016_DARTFISH
    • Amount: 341ng
    • Physical State: Solid
  • Calculate MW of 195 nt probe = 195nt * 303.7Da/nt + 79Da = 59,300.5 g/mol
  • 341ng -> 5.75pmol

Resuspend and Aliquot[edit]

  • Resuspend in 100ul (57.5nM)
    • Pipetted 20X to mix
  • Made aliquots of 40, 40, 10, and 10 into 0.5ml Lo-Bind tubes

Serial Dilution 1000X[edit]

  • Take 1ul from a 10ul aliquot and add 9ul H2O (10X Dilution)
  • Take 5ul and add 45ul H2O (100X Dilution)
  • Take 20ul and add 180ul H2O (1000X Dilution)

Expansion PCR Test[edit]

  • NTC
Component Volume
Seed oligo 0
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 10.5
Total 25
  • Positive Control
Component Volume
CA12kNov2014_V4 1st rnd Oligos (10nM) 2.5
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 8
Total 25
  • 1,000X Dilution
Component Volume
Seed oligo (100X Dilution) 2.5
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 8
Total 25
  • 10,000X Dilution
Component Volume
Seed oligo (1000X Dilution) 2.5
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 8
Total 25

Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 40 -> 72C 2min -> 15C hold

File:20160526 TB12k ExpansionPCRtest.JPG

  • 1,000X final dilution was in the sweet spot for number of cycles (15-20)
  • Use that concentration and do 19 cycles

Expansion PCR[edit]

Component Volume
Seed oligo (100X Dilution) 15
F/R V4 Primer Mix (10uM) 12
2X Kapa MM 75
H2O 48
Total 150
  • 50ul per PCR tube

Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold

  • Purified with 2 Qiagen PCR columns (75ul PCR product each)
    • Elute with 50ul H2O
  • Quantify with Qubit dsDNA:
    • 10.1ng/ul / (195bp*607.4Da/bp+157.9Da) = 85.16 nmol/L
  • Dilute to 10nM
    • 85.16nM x 99ul = 10nM x 843ul
    • Add 744ul H2O

Production PCR[edit]

  • V4 master mix
Components Volume (1X) Volume (50X)
First round amplicon TB12k_Apr2016_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

Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold

  • Since Biorad qPCR can only do 50ul had to split into 2 96-well reactions
  • First stopped after 14 cycles

File:TB12k Apr2016 V4 ProductionPCR 14cycles.JPG

  • Second stopped after 12 cycles

File:TB12k Apr2016 V4 ProductionPCR 12cycles.JPG

  • In the future can do 14 cycles

EtOH Precipitation[edit]

  • 8 15-ml tubes (with 24 wells (3 strips) of PCR product each) for
    • 1,200ul PCR product
    • 3000ul 100% EtOH
    • 4ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 30 min
  • 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

Qia Column Purification[edit]

  • 8 columns elute 50ul each
  • Nanodrop: 287.3 ng/uL x 400uL = 114.9ug

Lambda Exo[edit]

  • Divide into 8 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 2hr (normally 1hr)
  • Purified with 8 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 91.1ng/ul x 320ul = 29.15ug (50.7% yield)

Remove Amplification Adapters[edit]

USER[edit]

  • Split into 5 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 64
USER 5
10X DpnII Buffer 8
H2O 3
Total 80
  • Incubate at 37C for 2.5 hours

DpnII[edit]

  • 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[edit]

  • Eluted 20ul each column (100ul total)
  • Nanodrop
    • 147.2 ng/ul x 100ul = 14.7ug (50.4% yield)

PAGE Size Selection[edit]

  • Run 4 gels
  • 200V for 40min
Components 4X Volume
V4 Probes 95
TBE-Urea Buffer 2X 95
Components 4X Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40

File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel1.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel2.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel3.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel4.jpg

EtOH Precipitation[edit]

  • Put cut out gel in 8 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 (~3500ul)
  • Precipitated in 8 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 8 tubes at -80C for 30min
  • Spun 8 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[edit]

20.2ng/ul x 74ul = 1,494.8ng 20.2ng/ul / (157nt x 303.7Da/nt + 79Da) = 423nM