Making electrocompetent cells protocol
- Modified for general electrocompetent cell prep (not specifically for recombineering): PMID: 19180090
- Pick an isolated colony from an LB plate and grow overnight in 3–5 ml of LB at 37C.
- Next morning, add 0.5 ml of the culture to 25 ml of LB in a 250-ml flask and grow at 37C to an OD600 of 0.50–0.60. Transfer the culture to a 50-ml Falcon tube and spin at 6,000g in prechilled rotor for 10 min at 4C.
- Wash the cell pellet with 20 ml of ice-cold H2O once and then resuspend in 1 ml of H2O and transfer to a chilled 1.5-ml tube. Spin at 10,000g for 20–30 s at 4C.
- Wash the cells two more times with 1 ml of ice cold H2O. Resuspend the cell pellet in H2O in a final volume of 100μl and keep on ice.
- Mix 100 ng of DNA with 50μl of electrocompetent cells and chill on ice for 5 min then transfer into a 0.1-cm cuvette. Introduce the DNA into the cells by electroporation (1.8 kV, 25 mF capacitance and 200 O resistance). Also do a control electroporation where no DNA is added. After electroporation, immediately add 1 ml of SOC and transfer to 1.5mL microcentrigure tube. Incubate cells (with shaking or rotation) at 37C for 1 h. Spin down cells for 2 min at 4000g in a microcentrifuge. Resuspend the pellets in 200 μl of LB. Plate each aliquot of cells on a single LB plate containing the appropriate antibiotic. Grow for 12-16 hours (overnight) at 37C.
First day of class outline
adapted from MIT 20.109
- Pipetting (micropipettes and pipetaids) - guided
- Make dilutions into cuvettes
- Making Solutions (pH meter) - self guided
- Spectrophotometer (and nanodrop) - self guided
- Sterile Technique - guided
- Setting up your work area
- Maintaining a clean environment - EtOH bench, pipets
- Working with sterile solutions - using flame
- Lab Math (dilutions etc)- self guided
- Lab Safety - self guided
- waste disposal - biohazard, glass, chemical
- safety stations - eye wash, shower
- stuff to be careful about - EtBr, using transilluminator
2nd day outline
- quiz over whatever reading was assigned
- figure out what they should read - something about transformations, plasmids, antibiotic resistance.... making electrocompetant cells
- tell them beforehand that theyll be getting a plasmid so they can start figuring out what to do with it.. how to figure out what it is doing.
- everybody gets a plasmid - maybe measure concentration and make a dilution? because only need a tiny amount of plasmid for transformation..
- Make electrocompetent cells
- transform plasmid in by electroporation
- plate on amp and kan plates plus controls
- come in next day to look at plates and take them out
- tell them to think about how transfer curves, inducible promotors?
- have the come in the night before to inoculate cultures to do minipreps on tuesday.
- have vectors prepped already
- transformation (make e-comp cells again..)
At each bench (2 people per bench):
- 2 P20
- 2 P200
- 2 P1000
- 1 P2
- 1 pipetaid
- 2 timers
- 2 microcentrifuge racks
- 1 culture tube rack
- 1 combo rack
- freezer box
- ice bucket
- Tips (2/20, 200, 1000)
- Autoclaved microcentrifuge tubes
- autoclaved glass beads
- kim wipes
- Squirt/Spray bottle with 70%EtOH
- Waste containers
- sterile water (100ml)
- LB Media (+antibiotics) - 250ml (keep in fridge?)
- aliquot kit buffers?
At each bay (for 2 pairs of people):
- Table top centrifuge
- bunsen burner
- Waste containers
- glass beads
- kit waste
Stuff that needs to be made/done for the first week
For the first day:
- write instructions for sterile station, lab safety station; add nanodrop stuff to spec station
- 0.01% xylene cyanol (take a little bit from lab..)
- order sorbitol (or figure out something else to use)
- order spec cuvettes
- order glassware (need to figure out what glassware we need)
- wash and label new glassware
- organize new glassware
- order more pipetaids
- order lab tape?
- order pipette tips (all size, double check what we have available)
- order big pipette things
- order more spatulas, stir bars - designate dirty spatula & stir bar container
- make water squeeze bottle for pH station
- make 70% etoh bottles for each bench (and sterile technqiue station)
- set up aspirators
- prep somekind of plasmid to use on nanodrop
- organize the lab and label where stuff goes
- set up each bench, write inventory sheet for students to go thru on first day?
For the second day:
- LB - 250mL bottles -> 8 bottles (2L total)
- sterile water - 100mL bottles -> 8 bottles?
- LB agar plates - amp and kan, at least one sleeve each
- antibiotic stocks? 10mL of each?
- autoclave eppis
- autoclave beads
- prep all plasmids, aliquot?
- try out making electocompetent cells
- order ep cuvettes