Running a DNA Gel
- The wax was removed from the PCR product.
- 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
- Loading Order: ladder - skip - M33S Hemoglobin - skip - experimental PCR product - skip - neg. control PCR product
- Run at 100V until dye has moved approximately 3/4 down the gel
Digestion of Wild-type DNA
- 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
- Take a sterile eppendorf tube and place it on ice.
- Mix 50μL of NovaBlue Competent E.coli with 5μL of the PCR product in the cold, sterile tube.
- Incubate this mixture for 30 minutes on ice.
- Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
- Add 200μL of SOC media to the cells/PCR product.
- Shake the mixture at 250rpm at 37°C for one hour.
- 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.
- Incubate the plate overnight (inverted) at 37°C.
Update 1/28/12: lots of colonies grew overnight on the plate