Daniel:Notebook/PosSequencing/2017-5-8

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Probe Production (Started Friday May 5)[edit]

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Protocol[edit]

After the success on Friday, I want to redo the production protocol but for the EcoRI primer. This makes it so that I can mix the two in relative quantities and test out the USER cleavage protocol.

  1. qPCR
    1. Make 4.2X (1X) master mix with the following reagents
      1. 4.2 (1) uL 10 uM AP1v4U
      2. 4.2 (1) uL 10 uM BiotinEcoRI
      3. 75.6 (18) uL nfH2O
      4. 105 (25) uL 2X SYBR Fast
    2. Add 45 uL master mix to each sample
    3. Add 5 uL barcode oligo according to plate layout
    4. File:20170505-PlateLayout-BiotinOligoProduction.png
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  2. Qiaquick Column
    1. Add 250 PB (5X binding buffer) to 50 sample
    2. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    3. Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
    4. Dry spin column for 1 minute at 14000 rpm; discard flow through
    5. Let stand with cover open in fume hood for ~5 minutes
    6. Transfer column to a new 1.5 mL eppendorf tube
    7. Add 35 uL nfH2O to column
    8. Let stand 1 minute
    9. Spin for 1 minute at 14000 rpm
  3. TBE Gel
    1. Mix 48 uL TBE and 12 uL 6X dye
    2. Aliquot 10 uL mix to parafilm
    3. Add 2 uL sample or 1.5 uL ladder to appropriate aliquot
    4. Add 10 uL mix to appropriate lane
    5. Run gel at 240V for 24 minutes
    6. Open gel and stain with 2 uL SYBR gold for 3 minutes
    7. Rinse and image in gel doc
  4. Qubit
    1. Mix 1194 uL dsDNA buffer and 6 uL dye
    2. Aliquot 190 uL to standard tubes and 199 uL to dye tubes
    3. Add 10 uL standard or 1 uL dye
    4. Vortex and spin down
    5. Let sit 5 minutes in the dark
    6. Measure in the Qubit

Results[edit]

Sample Qubit Conc (ug/mL) Amount (nM) Total Amount (fmol) Amplification (Over 5 amol)
Barcode Oligo v1 9.3 300 10493 2098646
Barcode Oligo v2 11.9 384 13427 2685364
Barcode Oligo v3 8.01 258 9038 1807544
NTC 0.15 5 169 33849

Bead Binding Test[edit]

Sample Matrix

Sample # Condition Emulsion Y/N
1 Barcode Oligo 1 N
2 Barcode Oligo 3 N
3 Barcode Oligo 1/3 Mix N


  1. Bead Binding
    1. Suspend 2 uL (20 ug, ~20M beads) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend in 20 uL Dynabuffer (conc. 1 ug/uL)
    4. Add 8 uL single barcode oligo or 4 uL each for sample 3 and up to 12 uL nfH2O per sample to bead solution; incubate at RT for 15 min
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    6. Repeat wash step
  2. Melting
    1. Resuspend in 100μL of TE-Triton 0.1% (TE-T)
    2. Melt off the complementary DNA strand by incubating the sample at 95°C for 3 minutes
    3. Magnet pull-down for 1 minute and remove supernatant
    4. Add 100 μL of TE-T and mix
    5. Repeat wash
  3. Fluorescent Oligo Binding (All samples)
    1. Resuspend beads in 25 uL 4X SSC buffer
    2. Make the following 3.1X (1X) dye hybridization buffer
      1. 6.2 (2 ea; 6 total) each uL 10 uM dye; dcprobe6-488, dcprobe6-Cy3, dcprobe6-Cy5
      2. 46.5 (15) uL formamide
      3. 12.4 (4) uL nfH2O
    3. Incubate probes at 80C for 5 minutes
    4. Add 25 uL fluorescent probe in 60% formamide to each sample
    5. Incubate at room temp in the dark for 30 min
    6. Wash twice in 2X SSC
    7. Resuspend in 40 uL 2XSSC or 20 uL 2X SSC