Experiments
Highlights: August 26th, 2017
In order to continue our experiments in using E. coli to insert plasmids, we first had to confirm if it was competent.
Competent Cell Testing
Purpose — to test if E. coli can take up plasmids
Optimization and Troubleshooting — The transfection was unsuccessful because, when the cultures didn’t grow for the first trial, we tried restreaking to see if anything would grow. However, this contaminated the plates, which is most likely the reason the transfection didn’t work. We plan to look for another medium to use.
Procedure:
- Sterilized working area with 70% ethanol.
- Thawed competent cells on ice. Labelled one 1.5 mL microcentrifuge tube for each transformation and then placed tubes on ice. Did triplicates of each concentration.
- Centrifugated the DNA tubes from the Competent Cell Test Kit to collect all of the DNA into the bottom of each tube. Adjusted settings to 20-30 seconds at 8,000-10,000 rpm.
- Pipetted 1 µL of DNA into each microcentrifuge tube.
- Pipetted 50 µL of competent cells into each tube. Flicked tube gently with your finger to mix.
- Incubated the cells on ice for 30 minutes. While waiting, pre-heated waterbath now to 42°C.
- Heat-shocked the cells by placing into the waterbath for 45 seconds.
- Immediately transfered the tubes back to ice, and incubated on ice for 5 minutes.
- Added 950 µL of SOC media per tube, and incubated at 37°C for 1 hour shaking at 200-300rpm. While waiting, prepared the agar plates (labelled them).
- Pipetted 100 µL from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Used sterile glass beads to help spread the mixture.
- Incubated at 37°C overnight or approximately 16 hours. Positioned the plates with the agar side at the top, and the lid at the bottom.
- Counted the number of colonies on a light field or a dark background, such as a lab bench. Used the average cell colony count from all three plates (triplicates) in the calculation.
Highlights: August 19th, 2017
We first grew subcultures of E. coli to use when testing for its competency in a future experiment. In addition, we familiarized ourselves with autoclaving and learned how to store E. coli for future reference.
Creating Subcultures
Purpose — to grow a culture of E. coli
Equipment and reagents — LB Broth Miller (autoclaved), competent cells, resuspended DNA, control DNA, SOC media, petri dishes, ice, pipette, tubes, water bath, shaker, spreader
Protocol:
1. Sterilized work area and all bottles and equipment with 70% ethanol.
2. Pipetted 1000μL of LB media into a tube.
3. Placed a sample of E. coli (Catherine’s sample) into a bucket of liquid nitrogen.
4. Used a syringe to obtain a small piece of the e. coli and swirl with the LB media.
5. Loosened the cap and incubated at 37°C.
Autoclaving
Purpose — to familiarize ourselves with the autoclaving process
Equipment — autoclave machine, tray, water, bottle of liquid
Protocol:
1. Obtained a tray and filled with a small volume of water.
2. Placed bottle on tray. Loosened cap and stuck indicator tape on the lid.
3. Placed in autoclave machine and adjusted settings.
Storing E. coli
Purpose — to learn how to store E. coli for future experiments
Equipment and reagents — glycerol, sample tube, LB Broth Miller (autoclaved), liquid nitrogen
Protocol:
1. Added glycerol to sample tube so that the ratio is 1 part media to 1 part glycerol. Final concentration of glycerol should be 25%.
2. Dropped in bucket of liquid nitrogen.
3. Stored in -80°C.
Source: http://parts.igem.org/
Safety
