User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/02/10

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Making 10mL TE Buffer

  1. Combine 20μL 0.5M EDTA with 100μL 1M Tris, pH 7.5 and 9.89mL distilled H2O.
    • 0.5M EDTA (1mL) = 0.146g EDTA + 1mL distilled water
    • 1M Tris, pH 7.5 was previously made and stored at 4°C.
  2. Sterile filter the TE buffer.


DNA extraction from Filter Paper

  1. Cut circle with DNA (wt swMb pT7-7) out of filter paper with a sterile blade.
  2. Place circle into sterile 1.5mL microcentrifuge tube.
  3. Add 30μL of sterile TE buffer to the tube.
  4. Vortex the tube to mix, and then spin the tube so that the paper and buffer are both at the bottom of the tube.
  5. Let sit for approximately a half hour at room temperature.

Transformation

  1. Take a sterile eppendorf tube and place it on ice.
  2. Mix 200μL of NovaBlue Competent E.coli with 5μL of the TE buffer mixed with the filter paper DNA.
  3. Incubate this mixture for 30 minutes on ice.
  4. Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
  5. Add 800μL of SOC media to the cells/PCR product.
  6. Shake the mixture at 250rpm at 37°C for one hour.
  7. Spread 100μL of the mixture on an LB plate with 100μg/mL ampicillin.
    • The LB plate was made with 0.875g LB, 0.7g Agar, and 35mL of distilled water. The mixture was autoclaved on a liquid cycle. When the mixture cooled to about 60°C 35μL of 100mg/mL ampicillin was added to it and it was poured into a sterile petri dish.
  8. Incubate the plate overnight (inverted) at 37°C.



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