Experiments
Protocols
Transformation
- Thaw 100 microliters of competent cells on ice.
- Add 5 microliters of DNA (plasmids) to the thawed competent cells.
- Incubate the cells and DNA on ice for 30 minutes.
- Place the tubes with the cells and DNA in a 42 deg. Celsius bath for 45-60 seconds.
- Leave cells on ice for about 2-5 minutes.
- Add 450 microliters of LB media to the cells.
- Pellet out the cell by centrifuging at 6000 RPM for 60 seconds.
- Remove 350 microliters of the supernatant and resuspend the cells.
- Pipet 100 microliters of the cells onto the appropriate plate and spread the cells throughout the plate using inoculation loops
Preparation of LB Media
- 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).
- Add 10 g/L of Tryptone.
- Add 10 g/L of NaCl.
- Add 5 g/L of Yeast Extract.
- Stir and fill up remaining volume with distilled water.
Preparation of LB Agar
- 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).
- Add 10 g/L of Tryptone.
- Add 10 g/L of NaCl.
- Add 5 g/L of Yeast Extract.
- Add 20g/L of Agar.
- Stir and fill up remaining volume with distilled water.
Preparation of Competent Cells
- Thaw 50 microliters of cells on ice for 5 minutes.
- Fill an overnight culture tube with 5 mL of LB media.
- Touch the tip of a pipet to the appropriate cells (either in a tube or on a plate) and place the cells into the culture tube.
- Grow the culture overnight for 16-18 hours at 37 deg. Celsius.
- During the next day, add 100 microliters of culture and 900 microliters of LB media to a cuvette.
- Measure the optical density (OD) of the sample using a spectrophotometer.
- Dilute the culture with LB media so that the OD of a sample is about 0.08.
- Incubate the culture at 37 deg. Celsius for about 75 minutes.
- Measure the OD of a sample by diluting by two and doubling the reading given.
- Incubate the culture at 37 deg. Celsius for 15-30 minutes (depending on the OD measured).
- Repeat the previous two steps until the OD lies between 0.4 and 0.6.
- Split the culture into 50 mL centrifuge tubes and place them on ice.
- Spin the tubes at 3000 RPM for 10 minutes at 4 deg. Celsius.
- Resuspend each pellet of competent cells in 5 mL of TSS buffer and place the tubes back on ice.
- Aliquot 100 microliters of the cell solution into separate microcentrifuge tubes while they are on ice.
- Place the microcentrifuge tubes of cells into the -80 deg. Celsius storage.
Preparation of LB + Ampicillin (Amp) Plates
- Measure 100 mg of solid Ampicillin per mL of antibiotic intended to be made.
- Dissolve the Ampicillin with distilled water and fill to the volume intended.
- Heat previously prepared LB agar so that it has completely melted.
- Filter the Ampicillin solution through a syringe with an appropriate filtration tip.
- Add Ampicillin solution to the LB agar so that there is 1 microliter of Ampicillin solution per 1 mL of LB agar.
- Add about 20 mL of the LB + Amp mixture into each empty plate.
- Let the LB + Amp cool and harden and then store the plates at 4 deg. Celsius.
Preparation of LB + Chloramphenicol (CM) Plates
- Measure 30 mg of solid Chloramphenicol per mL of antibiotic intended to be made.
- Dissolve the Chloramphenicol with distilled water and fill to the volume intended.
- Heat previously prepared LB agar so that it has completely melted.
- Filter the Chloramphenicol solution through a syringe with an appropriate filtration tip.
- Add CM solution to the LB agar so that there is 1 microliter of CM solution per 1 mL of LB agar.
- Add about 20 mL of the LB + CM mixture into each empty plate.
- Let the LB + CM cool and harden and then store the plates at 4 deg. Celsius.
Miniprep
- Incubate a cell culture overnight at 37 deg. Celsius.
- Transfer the culture to microcentrifuge tubes.
- Centrifuge the microcentrifuge tubes at 3000 RPM for 2 minutes and decant the supernatant.
- Resuspend the cell pellets in 250 microliters of resuspension solution.
- Add 250 microliters of lysis buffer.
- Add 350 microliters of neutralization solution and mix immediately.
- Centrifuge the tubes at 10,000 RPM for 15 minutes.
- Transfer to spin columns with microcentrifuges attached.
- Centrifuge for 1 minute at 10,000 RPM and discard the flow-through.
- Add 500 microliters of wash solution into the spin column.
- Centrifuge for 30-60 seconds at 10,000 RPM and discard flow-through
- Repeat the previous two steps.
- Centrifuge for 1 minute at 10,000 RPM.
- Transfer the spin columns onto new 1.5-mL microcentrifuge tubes and add 50 microliters of elution buffer.
- Incubate for 2 minutes at room temperature.
- Centrifuge for 2 minutes at 10,000 RPM.
- Discard the spin column and store the DNA solution in the microcentrifuge tube at -20 deg. Celsius.
Restriction Digestion
- The volume of DNA solution depends on its concentration.
Gel Electrophoresis
- Mix 50 mL of TAE (Tris-acetate-EDTA) buffer with a certain mass of agarose so that the mass concentration is 1-3%