Daniel:Notebook/PosSequencing/2017-6-1

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Emulsion PCR Test 3 (Started Yesterday)[edit]

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Protocol (Continued from Yesterday)[edit]

  1. Emulsion Breaking
    1. Set aside 15 uL for qPCR
    2. With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside
    3. Add 100 uL isopropyl alcohol (IPA) to tube with 200 uL tip (use new tip)
    4. Draw up IPA with tip from part A, mix to resuspend tip
    5. Vortex the pooled sample in the 1.5 mL tube for 30 seconds
    6. Spin down the sample 13,000 rpm for 5 minutes
    7. Take off oil-IPA mix (top layer) but do not disrupt the oil/aqueous interface
    8. Add 1000 uL IPA and vortex for 30 seconds
    9. Spin down sample at 13,000rpm for 3 minutes
    10. Again remove IPA without disturbing the pellet
    11. Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
    12. Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend
    13. Leave on 1.5mL mixer at 1300rpm 1 minute
    14. Spin down sample at 13,000rpm for 1 minute
    15. Remove all but 40uL wash buffer
    16. Repeat steps 11 to 15 two times (j to o)
    17. On last wash, take 15 uL (of 100) for qPCR
    ******
    
  2. Strand Separation (DNA Denaturation)
    1. Add 500 uL KOH buffer (pH 12.0) and leave on 1.5mL mixer at 1300rpm 1 minute
    2. Spin down sample at 13,000rpm for 1 minute
    3. Remove all but 40uL buffer
    4. Repeat KOH steps A-C once more
    5. Add 500 uL 4X SSC buffer (pH 12.0) and leave on 1.5mL mixer at 1300rpm 1 minute
    6. Spin down sample at 13,000rpm for 3 minutes
    7. Remove all but 10uL buffer
    8. Resuspend in 80 uL 2X SSC buffer
    9. Use this for final step of qPCR
    ******
    
  3. Single Primer qPCR
    1. Make the following master mixes
    2. Reagent uL in Single Rxn Master Mix P2 (9.2X) Master Mix BioU (9.2X) Master Mix Dual (5.2X)
      Primer 2 (10 uM) 1 9.2 0 5.2
      BioU Primer (10 uM) 1 0 9.2 5.2
      2X Kapa SYBR Master Mix 20 184 184 104
      nfH2O 17 156.4 156.4 83.2
      Total* (+ 2 uL sample=40) 38 349.6 349.6 197.6
    3. Aliquot 38 uL master mix to appropriate lanes
    4. Add 2 uL sample according to plate layout below
    5. File:PlateLayout-20170601-SPqPCR-emPCRTest3.png
    6. Run the following thermocycler program
      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

Results[edit]


Conclusions:

  • I need to run this with more cycles; the dual Aqueous and ssDNA showed DNA with exponential amplification but not linear;
  • Given the number of cycles for exponential amplification there is not much
  • Oil gives no signal (too messy)
  • The normalized values show a little better that the only real signal was the dsDNA samples amplified with single primers

Backup on the qPCR log. I'll run this tomorrow