Making 10mL TE Buffer
- 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.
- Sterile filter the TE buffer.
- Cut circle with DNA (wt swMb pT7-7) out of filter paper with a sterile blade.
- Place circle into sterile 1.5mL microcentrifuge tube.
- Add 30μL of sterile TE buffer to the tube.
- Vortex the tube to mix, and then spin the tube so that the paper and buffer are both at the bottom of the tube.
- Let sit for approximately a half hour at room temperature.
Transformation
- Take a sterile eppendorf tube and place it on ice.
- Mix 200μL of NovaBlue Competent E.coli with 5μL of the TE buffer mixed with the filter paper DNA.
- 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 800μL 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.
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