Matt:LabNotes/2016-6-20

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TB12k_Apr2016 V7 Mouse Brain Probe Preparation[edit]

Expansion PCR[edit]

Component Volume
Seed oligo (100X Dilution) 15
F/R V7 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
File:2016-06-20 TB12kApr2016 V7 ExpansionPCR.jpg

  • Purified with 3 Qiagen PCR columns (50ul PCR product each)
    • Elute with 50ul H2O
  • Quantify with Qubit dsDNA:
    • 9.2ng/ul / (195bp*607.4Da/bp+157.9Da) = 77.57 nmol/L
  • Dilute to 10nM
    • 77.57nM x 144ul = 10nM x 1117ul
    • Add 973ul H2O

Production PCR[edit]

  • V7 master mix
Components Volume (1X) Volume (50X)
First round amplicon TB12k_Apr2016_V7 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V7U (100uM) 0.4 20
AP2V7 (100uM) 0.4 20
H2O 49 2450
Total 100 5000

Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold
File:2016-06-20 TB12kApr2016 V7 ProductionPCR.jpg

EtOH Precipitation[edit]

  • 4 5-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]

  • 4 columns elute 50ul each
  • Nanodrop: 193.0 ng/uL x 200uL = 38.6ug

Lambda Exo[edit]

  • 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 1.5 hr (normally 1 hr)
  • Purified with 4 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 68.4ng/ul x 160ul = 10.944 ug (% yield)

Remove Amplification Adapters[edit]

USER[edit]

  • Split into 3 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 53
USER 5
10X DpnII Buffer 8
H2O 14
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_V7 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 (60ul total)
  • Nanodrop
    • 111.4ng/ul x 60ul = 6.684ug (62.5% yield)

PAGE Size Selection[edit]

  • 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:2016-06-23 TB12kApr2016V7 ProbeProduction SizeSelect.jpg

  • Only saw one faint band above main band that was cut out
    • That means either the USER or DpnII digestion was very complete or did not occur at all
    • I am sure I used the corrent DpnII-V7 oligo
  • Can run a gel comparing with 1st round amplicons to check for size next time

EtOH Precipitation[edit]

  • 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 4 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 450ul 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 8 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[edit]

18.2ng/ul x 40ul = 728ng 18.2ng/ul / (160nt x 303.7Da/nt + 79Da) = 374 nM