ASIJPROTOCOL
Protocol
Competent Cell Check
- Spin down the DNA tubes from the Competent Cell Test Kit/Transformation Efficiency Kit to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 rpm will be sufficient.
- Note: There should be 50 µL of DNA in each tube sent in the Kit.
- Thaw competent cells on ice. Label one 2.0ml microcentrifuge tube for each concentration and then pre-chill by placing the tubes on ice.
- Pipet 1 µL of DNA into each microcentrifuge tube. For each concentration, use a separate tube.
- Pipet 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.
- Incubate on ice for 30 minutes.
- Pre-heat waterbath now to 42°C.
- Heat-shock the cells by placing them into the waterbath for one minute. Be careful to keep the lids of the tubes above the water level, and keep the ice close by.
- Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover.
- Add 200 µL of SOC media per tube, and incubate at 37°C for 2 hours. Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture.
- Pipet 20 µL from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Do triplicates (3 each) of each tube if possible, so you can calculate an average colony yield.
- Incubate at 37°C overnight or approximately 16 hours. Position the plates so the agar side is facing up, and the lid is facing down.
- Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency. If you've done triplicates of each sample, use the average cell colony count in the calculation. Make sure to measure the area of each colony to see how effective our cells are.
Gibson Assembly
Purpose: Design primers to amplify fragments (and/or vector) with appropriate overlaps PCR amplify fragments using a high-fidelity DNA polymerase.
- Prepare linearized vector by PCR amplification using a high-fidelity DNA polymerase or by restriction digestion.
- Confirm and determine concentration of fragments and linearized vector using agarose gel electrophoresis, a NanoDrop™ instrument or other method.
- Add fragments and linearized vector to Gibson Assembly Master Mix and incubate at 50°C for 15 minutes to 1 hour, depending on number of fragments being assembled.
- Transform into NEB 5-alpha Competent E. coli (provided) or use directly in other applications.
PCR Protocol
- Combine 10 μl of dnTP, 5 μl of PCR buffer, 2 μl of DNA polymerase, 10 μl of DNA template, 5 μl of forward primer, and 5 μl of reverse primer into one tube.
- Mix tube by tapping it on the table.
- Put it into the thermocycler and run it for 30 cycles.
e. Coli PET Plastic Degradation Protocol
- Weigh out the same mass of PET plastic to put into each e. Coli colony.
- Will have one control and one colony for each of the promoter strengths.
