Daniel:Notebook/ComboLock/2017-3-21

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Revision as of 16:32, 21 March 2017 by >Djacobse (→‎Bgl Digestion)
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Circularization Comparison (T4 Poly/Ligase; Started Wednesday)

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Bgl Digestion

Because I CAN. Also, will determine if there is an improvement in signal post-digestion.

Sample Matrix

Sample Origin Condition Sample Numbers
F T4 Ligase 1,4
G Phusion/Amp (Unpurified) 1,4
H Phusion/AmpHS (Unpurified) 1,4
  1. Bglii Digestion
    1. Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
    2. Make the following 4.2X master mix
    3. Reagent Single Rxn Vol 14.2X MM uL
      RCA Reaction Product 5 0
      Bglii Cutter Primer (10 uM) 5 71
      10X Buffer 3.1 2 28.4
      Bglii 1 14.2
      nfH2O 7 99.4
      Total 20 198.8
    4. Aliquot 14 uL master mix into new tubes
    5. Add 5 uL appropriate sample to each tube
    6. Use the following thermocycler program
      1. 5 min 95C
      2. 10 min 50C
      3. 1 hr 37C; When this cycle starts add 1 uL Bglii
      4. Heat kill with 65C for 20 min