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
    1. Make dilutions into cuvettes
  • Making Solutions (pH meter) - self guided
  • Spectrophotometer (and nanodrop) - self guided
  • Sterile Technique - guided
    1. Setting up your work area
    2. Maintaining a clean environment - EtOH bench, pipets
    3. Working with sterile solutions - using flame
    4. Aspirating
  • Lab Math (dilutions etc)- self guided
  • Lab Safety - self guided
    1. waste disposal - biohazard, glass, chemical
    2. safety stations - eye wash, shower
    3. 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



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