Daniel:Notebook/ComboLock/2017-3-16

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Revision as of 19:51, 16 March 2017 by >Djacobse (→‎Protocol-Part 4)
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Circularization Comparison (T4 Poly/Ligase)

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Protocol-Part 4

  1. Exonuclease Digestion
    1. Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1.5 hours at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. qPCR
    1. Make qPCR master mixes according to following recipe
    2. Reagent Single Rxn MMProd (15.2X) MM+ (C1, 4.2X)
      Primer Pair (fwd/rev) NA primer12*/AmpR-Ind21 primer24/primer4*
      Forward Primer (10 uM) 1 15.2 4.2
      Reverse Primer (10 uM) 1 15.2 4.2
      nfH2O 21 319.2 88.2
      2X Kapa SYBR Fast 25 380 105
      Total 48 729.6 201.6
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. File:PlateLayout-20170316-T4Circ-PreRCATest.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
  3. Padlock Hybridization-Y Samples
    1. Bake samples at 95 C and vortex to resuspend
    2. Make the following master mix
      1. 4.2 uL Padlock0601 (10 uM)
      2. 8.4 uL Amp Ligase 10X buffer
      3. 50.4 uL nfH2O
    3. Add 15 uL master mix to appropriate tubes
    4. Add 5 uL sample according to yesterday's tables
    5. Incubate at 50C for 20 minutes
    ******
    
  4. Circularization
    1. Make the following master mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (4.5x) (uL)
      NAD+ 5 mM 40 nmol 8 36
      dNTP 1 mM 600 pmol 0.6 2.7
      Betaine 5 M 15 umol 3 13.5
      10X AmpLigase Buffer 10X 1X 2 9
      Amp Ligase 5 U/uL 10 U 2 9
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 14.4
      nf H2O     1.2 5.4
      Total     20 90
    3. Follow the workflow in yesterday's diagrams