Experiments

Protocols

Transformation

1. Thaw 100 microliters of competent cells on ice.
2. Add 5 microliters of DNA (plasmids) to the thawed competent cells.
3. Incubate the cells and DNA on ice for 30 minutes.
4. Place the tubes with the cells and DNA in a 42 deg. Celsius bath for 45-60 seconds.
5. Leave cells on ice for about 2-5 minutes.
6. Add 450 microliters of LB media to the cells.
7. Pellet out the cell by centrifuging at 6000 RPM for 60 seconds.
8. Remove 350 microliters of the supernatant and resuspend the cells.
9. Pipet 100 microliters of the cells onto the appropriate plate and spread the cells throughout the plate using inoculation loops

Preparation of LB Media

1. Fill a container to about 70% of its volume with distilled water(assuming the container volume is the total volume of media intended to be made).
2. Add 10 g/L of Tryptone.
3. Add 10 g/L of NaCl.
4. Add 5 g/L of Yeast Extract.
5. Stir and fill up remaining volume with distilled water.

Preparation of LB Agar

1. Fill a container to about 60%-70% of its volume with distilled water(assuming the container volume is the total volume of media intended to be made).
2. Add 10 g/L of Tryptone.
3. Add 10 g/L of NaCl.
4. Add 5 g/L of Yeast Extract.
5. Add 20g/L of Agar.
6. Stir and fill up remaining volume with distilled water.

Preparation of Competent Cells

1. Thaw 50 microliters of cells on ice for 5 minutes.