Daniel:Notebook/ComboLock/2017-6-15

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Antibody Detection Test-Positive Control (Started Yesterday)

Back to Calendar=

Protocol-Part 3

  1. Padlock Hybridization
    1. Add 20 uL of following 6.2X Master Mix
      1. 6.2 uL 10 uM Padlock0601
      2. 12.4 uL 10X Amp Ligase buffer
      3. 105.4 uL nfH2O
    2. Incubate at 70C for 5 min
    3. Incubate at 50C for 15 minutes
    4. Wash once with wash buffer*Do not wash sample 5
    5. Resuspend in 20 uL 1X Amp Ligase buffer
    ******
    
  2. Circularization
    1. For phusion samples, make the following master mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (10.5x) (uL)
      NAD+ 5 mM 40 nmol 8 84
      dNTP 1 mM 600 pmol 0.6 6.3
      Betaine 5 M 15 umol 3 31.5
      10X AmpLigase Buffer 10X 1X 2 21
      Amp Ligase 5 U/uL 10 U 2 21
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 33.6
      nf H2O     1.2 12.6
      Total     20 210
    3. Add 20 uL circularization mix to each sample
    4. Incubate at 55C for 2 hours
    5. Incubate at 95C for 5 minutes
    6. Hold at 16C