Daniel:Notebook/PosSequencing/2017-5-19

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Double Stranded Probe Production[edit]

Back to Calendar

I need to make more of these.

Protocol[edit]

  1. qPCR
    1. Make three 8.2X (1X) master mix with the following reagents
      1. 8.2 (1) uL 10 uM AP1v4U
      2. 8.2 (1) uL 10 uM BiotinU
      3. 172.2 (21) uL nfH2O
      4. 205 (25) uL 2X SYBR Fast
      5. 16.4 (2) appropriate barcode oligo (100 pM)
    2. Add 50 uL master mix to 8 lanes per sample
    3. File:PlateLayout-20170519-BarcodeOligoProduction.png
    4. 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
  3. Note: I will be pooling samples and double loading columns, yielding 4 samples per column (2 columns per barcode oligo, 6 columns total)
    1. Pool 2 samples and 500 uL PB (5X binding buffer)
    2. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    3. Add another 600 uL pooled sample to columnand spin for 1 minute at 14000 rpm; discard flow through
    4. Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
    5. Dry spin column for 1 minute at 14000 rpm; discard flow through
    6. Let stand with cover open in fume hood for ~5 minutes
    7. Transfer column to a new 1.5 mL eppendorf tube
    8. Add 35 uL nfH2O to column
    9. Let stand 1 minute
    10. Spin for 1 minute at 14000 rpm
    11. Pool samples when finished; concentrate if desired
  4. 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
  5. 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 40 amol)
Barcode Oligo v1 20.1 324 2.6E+04 6.5E+05
Barcode Oligo v2 16.3 263 2.1E+04 5.3E+05
Barcode Oligo v3 18 290 2.3E+04 5.8E+05

Exonuclease Digestion Test[edit]

I'm going to use lambda exonuclease to digest the second strand (it can't digest the biotin strand since it has a 5' biotin)

  1. Phosphorylation
    1. Set up 2 reactions using the following table
    2. Reagent Final Conc. uL Added
      dsDNA (0.3 pmol/uL) (< 300 pmol) 10
      Reaction Buffer 10X 1X 5
      ATP (10 mM) 1 mM 5
      T4 PNK (10 U/uL) 10 Units 1
      nfH2O NA 29
      Total   50
    3. Incubate at 37C for 30 minutes
    4. Incubate at 65C for 20 minutes
  2. Lambda Exonuclease Digestion
    1. Prepare 2 reactions according to following table
    2. Reagent Final Conc. uL Added
      Phosphorylated Mix (<2 ug) 20
      Reaction Buffer 10X 1X 5
      Lamda Exonuclease (10 U/uL) 2 U/uL 2
      nfH2O NA 23
      Total   50
    3. Incubate at 37C for 30 minutes
    4. Incubate at 80C for 10 minutes
    5. Hold at 12C

Continued tomorrow