20.109(S08):Prepare expression system (Day3)

Protocols

Part 1: prepare competent DE3 cells

1. Pick up two 3 mL tubes of DE3 cells. These cells should be in or close to the mid-log phase of growth, which is indicated by an OD₆₀₀ value of 04.-06.
2. Measure the OD₆₀₀ value of a 1:10 dilution of your cells. If the cells are not yet dense enough, return them to the rotary shaker in the incubator. Remember to balance your tubes! As a rule, your cells should double every 20-30 min.
3. Once your cells have reached the appropriate growth phase, spin down 4 tubes of ~ 1.5 mL each, aspirate the supernatants, and resuspend in an equal volume of ice-cold calcium chloride (100 mM).
4. Spin again for 1 min. The resultant pellets should occur as streaks down the side of the eppendorf tube, so be very carefule not to disturb the cells when aspirating.
5. This time, resuspend each tube in 100 μL of CaCl₂, then pool the tubes together.
6. Incubate on ice for 1 hour.
7. Meanwhile, label four eppendorfs (for three transformations and a no DNA control) and pre-chill them on ice. (You might label your mutant tubes as X#Z, where # is the residue number you are modifying, X is the original amino acid, and Z is the mutant acid. The example shown on Day 1 would be Y64D.)

Part 3: count mutant colonies

When you have a spare moment today, count the colonies that arose on each of your transformed XL1-Blue plates. Do the control samples have no colonies? Do the different mutations appear to have different efficiencies?