Transferring our bacteria from a solid culture to a liquid culture.
Step 1: Pick one colony from the chosen transformation plate using a 10uL micropipette tip.
Step 2: Place micropipette tip into an Eppendorf tube containing 100 uL of nuclease free water. To resuspend the colony.
Step 3: Add 5 mL of LB broth to a 15 mL Conical tube along with the appropriate antibiotics
Step 4: Take 50 uL of the nuclease-free water and colony mixture and add it into the 15 mL conical tube.
Step 5: Take the other 50 uL of the water and colony mixture and plate it on the appropriate antibiotic plate
Step 6: Store the overnights and plates on the 37C.
Step 7: Place all leftover water and colony mixture into the fridge. Labeled with the appropriate colony designation and the date
Glycerol Stock:
Step 1: Add 5 mL of LB Broth plus antibiotics to a 15 mL Conical Tube
Step 2: Using a 5 mL glass pipet, stab the glycerol stock and transfer a shard of the stock to the LB
Step 3: Incubate overnight at 37C
The purpose of plasmid preps is to isolate the plasmid DNA from the bacteria culture so we can perform a PCR. We used the Monarch Plasmid Miniprep Kit to do this.
Step 1: Pellet 1-5 ml (not to exceed 15 OD units) bacterial culture by centrifuging at 16000g for 30 seconds. Discard supernatant.
Step 2: Resuspend pellet in 200 uL of B1 Resuspension Buffer. Vortex to ensure suspension.
Step 3: Add 200 uL of B2 Plasmid Lysis Buffer and invert. Let sit at room temperature for one minute.
Step 4: Add 400 uL of B3 Neutralization Buffer, invert a few times, and let sit at room temperature.
Step 5: Centrifuge at 16000g for 3 minutes.
Step 6: Transfer supernatant to labeled spin column and centrifuge again at 16000g for one minute. Discard flow-through.
Step 7: Re-insert the column into the collection tube and add 200 uL of Plasmid Wash Buffer 1. Centrifuge at 16000g for one minute and discard flow-through.
Step 8: Add 400 uL of Plasmid Wash Buffer 2 and centrifuge for one minute.
Step 9: Transfer column to a clean 1.5 mL micro-centrifuge tube.
Step 10: Add 30 uL of DNA Elution Buffer to the center of the matrix, and then wait one minute before centrifuging at 16000g for one minute.
Step 11: Use the Nanodrop machine to check the concentration of DNA in the final eluted DNA.
Adapted from New England BioLabs
Once our constructs had been successfully assembled into our expression vectors, they next had to be transferred into our bacteria to successfully begin producing our compounds.
Step 1: Thaw 50ul of competent BL21DE3 cells and add 2uL of DNA into competent cells. Incubate on ice for 30 minutes.
Step 2: Heat shock mixture in heat-block at 42ºC for 30 seconds and put back on ice for 2 minutes.
Step 3: Preheat Super Optimal Media with Catabolite Repression (SOC) media to 37ºC. Add 950uL of SOC to a final volume of ~1000uL
Note: This step should be done in a sterile Biosafety Cabinet (BSC)
Step 4: Incubate at 37ºC for 60 minutes. During this incubation period there needs to be aeration of some kind. For this, cells were put on a shake plate at 300 rpm.
Step 5: Dry three LB plates with appropriate antibiotics in a 37C incubator. Plate 100 uL onto one plate and then plate 200 uL onto each of the remaining plates. Spread evenly around plate using a Colony Spreader.
Step 6: Incubate at 37ºC overnight