Daniel:Notebook/ComboLock/2017-3-6

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Padlock0601 Test (Started Wed March 1)[edit]

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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!)[edit]

  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