Daniel:Notebook/ComboLock/2017-3-23

From ZhangLabWiki
Revision as of 19:01, 23 March 2017 by >Djacobse (→‎Results)
Jump to navigation Jump to search

Circular Product Production (Started Yesterday)

Back to Calendar

Protocol

  1. qPCR
    1. Make qPCR master mix according to following recipes
    2. Reagent 1X Rxn MM-Amp (6.2X) MM-Prod (7.2X)
      Primer Pair NA AmpF/AmpR p24/p12RC
      Forward Primer (10 uM) 1 6.2 7.2
      Forward Primer (10 uM) 1 6.2 7.2
      Kapa SYBR Fast Master Mix (2X) 25 155 180
      nfH2O 21 130.2 151.2
      Total 48 297.6 345.6
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. File:PlateLayout-20170323-CPProduction.png
    6. 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. Qubit Measurement
    1. Add 995 uL buffer and 5 uL ssdye to tube
    2. Add 190 uL buffer and 10 uL correct standard to standard tubes
    3. Add 199 uL buffer and 1 uL sample to sample tubes
    4. Let sit 5-10 minutes in the dark
    5. Measure in Qubit
  3. TBE Gel
    1. Mix 120 uL TBE and 40 uL 6X dye
    2. Aliquot 10 uL per sample on to parafilm
    3. Add 2 uL sample or 1.5 uL ladder to aliquots
    4. Mix and add 10 uL per lane to gel
    5. Run gel at 230V for 24 minutes
    6. Add 2 uL SYBR gold and incubate on shaker for 3 minutes
    7. Rinse and image in gel doc

Results