User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/01/27

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Running a DNA Gel

  1. The wax was removed from the PCR product.
  2. 10μL of the PCR product was mixed with 2μL of 6x loading dye, and this was loaded into a 1.2% agarose gel.
    • 1.2% Agarose Gel: 0.3g agarose + 25mL of 1x TAE buffer
  3. Loading Order: ladder - skip - M33S Hemoglobin - skip - experimental PCR product - skip - neg. control PCR product
  4. Run at 100V until dye has moved approximately 3/4 down the gel

Photo (7).JPG

Digestion of Wild-type DNA

  1. 1μL of DpnI was added to the PCR product and it was put on a heat block at 37°C for one hour.

Transformation of PCR Product

  1. Take a sterile eppendorf tube and place it on ice.
  2. Mix 50μL of NovaBlue Competent E.coli with 5μL of the PCR product in the cold, sterile tube.
  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 200μL 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.

Update 1/28/12: lots of colonies grew overnight on the plate