Daniel:Notebook/ComboLock/2017-3-6

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Revision as of 00:18, 7 March 2017 by >Djacobse (→‎Protocol)
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Padlock0601 Test (Started Wed March 1)

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After reading some papers I'm going to give it one more go to see if I can't amplify the DNA from last week better. I'll use the method of largest amplification I know: most time using random hexamers as primers.

RCA Repeat (Maximum Power!)

  1. Maximal Rolling Circle Amplification
    1. Create master mix according to following table
    2. Reagent Single Rxn Vol 16.2X MM uL
      Template 4 0
      RCA Hexamer (1 mM) 0.5 8.1
      dNTP (1 mM) 5 81
      10X Buffer 2 32.4
      Phi29 1 0
      BSA (10 mg/mL) 0.4 6.48
      nfH2O 7.1 115.02
      Total 20 243
    3. Add 16 uL master mix to each tube
    4. Add 4 uL template according to table
    5. Incubate at 95 C for 5 minutes
    6. Lower to 55C for 15 minutes
    7. Lower to 30C for at least 2 minutes
    8. Add 1 uL Phi29 polymerase to each sample
    9. Incubate 15 hours at 30C
    10. Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C

Continued tomorrow