Our protocols are based on standard lab protocols used in the lab of our advisers unless otherwise stated. These protocols were subsequently modified if necessary based on our experience. A knowledge of basic lab techniques such as pippeting, plating, etc. are assumed and not covered as there are pretty comprehensive tutorials on the web for those.
A list of materials mentioned in the protocols which we used is also included at the end of the page.

Kit Protocols

1. Z-Competent E. Coli Transformation Kit for making competent cells for easy transformation. Modified to incubate cells on ice for 1 hour during transformation
2. Phusion High Fidelity PCR Kit for amplifying our inserts and cassettes.
3. TAQ DNA Polymerase for verifying our cloned constructs
4. Qiagen Mini-prep Kit for isolating our plasmids from the cells, to be transformed into expression strains.
5. Zymo Clean and Concentrator kit for cleaning up our digestion products before ligation.

Cloning Protocol

1. If necessary, first prepare the insert using PCR amplification. This was done to add the necessary restriction sites for cloning. The PCR protocol follows the kit protocol outlined above, except with our specific range of melting and annealing temperatures calculated using NEB's Tm calculator
2. Start digestion of vector and insert DNA using desired restriction enzyme manufacturer protocol. [NEB]
3. After 2 hours, add Phosphatase [CIP] according to manufacturer protocol to insert to prevent self-ligation
4. Purify and clean DNA using kit protocol. [Zymo Research]
5. Measure vector/insert concentration. [Nanodrop]
6. Divide the concentration by the length of the sequence and calculate ligation ratios of 1 vector to 3 insert. Mix the ratios according to the calculations, including T4 buffer and ligase.
7. Leave ligation products at room temperature for 1 hour.
8. Transform ligation products into competent cells using appropriate protocol. Incubate on ice for 1 hour if using Zymo competent cells
9. Plate cells and incubate at 37 degrees overnight. Check for colonies the next morning. If there are colonies present, inoculate 1-3 colonies into (selective) L.B. in the evening and culture overnight.

Gel Protocol

1. For 7x7 cm, 0.5cm thick gel, with 1.75% agarose Concentration.
2. Add 0.35 grams of agarose to 20ml of 1x buffer (TBE) Note: Place mark on container to keep track of liquid level using marker, so water can be added to bring the liquid back to original level
3. Place gel solution in microwave. Use low/medium, set timer for 5 minutes. Stop the oven every 30 seconds and swirl gently to suspend undissolved agarose.
4. >Once dissolved, set aside to cool (~60 degrees). Once solution is warm to touch, add 0.5ug/ml ethidium bromide. [1µl of 10mg/ml]
5. Pour into casting tray, remember to add in comb at the cathode side (black). Gel will solidify in ~10 mins
6. Divide the concentration by the length of the sequence and calculate ligation ratios of 1 vector to 3 insert. Mix the ratios according to the calculations, including T4 buffer and ligase.
7. Remove comb for solidified gel, remove casting gates and submerge gel beneath 2 to 6mm of 1x buffer.
8. Mix loading dye with PCR/digested products, load mixture into wells, together with 10µl of ladder
9. Run gel at 75V for ~1 hour. Adjust voltage accordingly if require faster or more distinct gels.

Dosage Curve

Time Lapse Protocol

Materials Used