Daniel:Notebook/ComboLock/2017-1-30

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RCA Test (Started Monday)[edit]

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Library Prep (Started Jan 26)[edit]

  1. Ethanol Precipitation
    1. Centrifuge for 30 minutes at 12500 rpm at 4C
    2. Remove supernatant and add 750 uL chilled 70% EtOH
    3. Centrifuge for 15 minutes at 12500 rpm at 4C
    4. Remove supernatant; dry in vacuum centrifuge for 15 minutes
    5. Resuspend in 20 uL H2O and consolidate samples
    6. Measure with Qbit
    7. Consolidate with samples from Thursday 1-26

DEJ20160126-CLv3-RCA02

Final Sample Matrix

Sample Sample Amt Pre/Post RCA AmpR Index
Sample 0 (AB) 1 pmol Pre 21
Sample 0X (AB) 1 pmol Post 25
Sample 2X (AB) 10 fmol Post 26
Sample 4X (AB) 100 amol Post 27
Sample 6C (AB) 1 amol Epi 22
Sample 6D (AB) 1 amol Epi 1:10 23

RCA Timing Test (Started Friday)[edit]

Because of the results from Saturday, I think I want to try Epicentre again for as long as possible. I'll do 12 hours with (as usual) one negative control to save up on epicentre enzyme.

  1. Rolling Circle Amplification
    1. Prepare master mix according to table below
    2. Reagent uL Added Master Mix (4.2X)
      Template 4 NA
      RCA Primer (10 uM) 2.5 10.5
      dNTP (1 mM) 2 8.4
      10X Buffer 2 8.4
      Phi29 1 NA
      nfH2O 8.5 35.7
      Total 20 63
    3. Add 14 uL master mix to each tube
    4. Add sample and to new reaction tubes according to following table
    5. Incubate at 30C for 12 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step

    Continued tomorrow