Daniel:Notebook/PosSequencing/2017-6-2

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Revision as of 22:08, 2 June 2017 by >Djacobse (→‎Protocol-Part 2)
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Emulsion PCR Test 3 (Started Wednesday)

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Protocol-Part 2

  1. Single Primer qPCR
    1. Make the following master mixes
    2. Reagent uL in Single Rxn Master Mix P2 (5.2X) Master Mix BioU (5.2X) Master Mix Dual (5.2X)
      Primer 2 (10 uM) 1 5.2 0 5.2
      BioU Primer (10 uM) 1 0 5.2 5.2
      2X Kapa SYBR Master Mix 20 104 104 104
      nfH2O 17 88.4 88.4 83.2
      Total* (+ 2 uL sample=40) 38 197.6 197.6 197.6
    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-Round2.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 x150
      7. 72C 2 min
      8. 16C hold

Results

Conclusions:

  • It didn't show well, but the H4 lane (dsDNA +++ control, dual primer) had a weird curve that started negative and went to zero quickly. It could have been poor baseline correction (platewise instead of by lane or something), in which case it actually CT'd 1st.