User:Isis Trenchard/Notebook/BioE44 Stuff/2010/03/19

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Making electrocompetent cells protocol

 * Modified for general electrocompetent cell prep (not specifically for recombineering): PMID: 19180090
 * 1) Pick an isolated colony from an LB plate and grow overnight in 3–5 ml of LB at 37C.
 * 2) 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.
 * 3) 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.
 * 4) 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.
 * 5) 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
 * Aspirating


 * 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?

3rd day

 * have the come in the night before to inoculate cultures to do minipreps on tuesday.
 * quiz?
 * miniprep
 * digestion
 * have vectors prepped already
 * ligation
 * transformation (make e-comp cells again..)

Inventory
At each bench (2 people per bench):
 * Tools:
 * 2 P20
 * 2 P200
 * 2 P1000
 * 1 P2
 * 1 pipetaid
 * 2 timers
 * Storage
 * 2 microcentrifuge racks
 * 1 culture tube rack
 * 1 combo rack
 * freezer box
 * ice bucket
 * Consumables
 * Tips (2/20, 200, 1000)
 * Autoclaved microcentrifuge tubes
 * autoclaved glass beads
 * kim wipes
 * Squirt/Spray bottle with 70%EtOH
 * Waste containers
 * Tip waste
 * trash can?
 * Stocks/Media
 * sterile water (100ml)
 * LB Media (+antibiotics) - 250ml (keep in fridge?)
 * aliquot kit buffers?

'''At each bay (for 2 pairs of people):
 * Equipment
 * Minispin
 * Table top centrifuge
 * bunsen burner
 * striker
 * vortexer
 * Waste containers
 * aspirator
 * glass beads
 * kit waste
 * Consumables
 * parafilm
 * papertowel pack

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?
 * SOC?
 * 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