DIYbio/BOSSlab/Notebook/2011/05/11

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Preflight

what's our goal

dry-run through transformation protocol w/ BBa_J04450 on card using Invivogen lyocomp cells

  • lyocomp 116 cells, e. coli k12 (?), non-pathogenic
  • hot water bath; 42C, heat caution
  • Ian: "The chemicals we'll be using amount to waste water (E. coli), cheese whey (tryptone), Vegemite (yeast extract & NaCl), tofu firming salts (MgCl2 and MgSO4), jelly (agar) and antibiotics and the concentrations are fairly low, so no special dangers - i.e. don't eat it. Cleanup can be city sewer, since all of these are things that are already found in waste water."

what are the risks today to us

  • trivial; just doing a dry run w/ the water baths

what are the risks today to the community

  • water

what waste are we gonna generate

  • hot water; combusted propane gas

Protocol

  1. Thaw competent cells (Lyocomp E. coli) on ice.
  2. During this time pre-chill sterile centrifuge tubes on ice.
  3. Gently tap competent cell containers to mix
  4. Aliquot bacterial suspension in pre-chilled tubes (100 µl per tube).
  5. To the aliquots add 1 µl of plasmid (RFP PUC19).
  6. Gently tap centrifuge tubes to mix
  7. Incubate 30 min on ice.
  8. During this time pre-heat water bath to 42°C (don’t use heating block).
  9. Incubate samples at 42°C in the water bath for 60s. Do not shake.
  10. Immediately chill samples on ice for 2 minutes.
  11. Add 0.95 ml of room temperature SOC.
  12. Plate 10 µl of bacterial suspension on antibiotic-containing agar plate. (filter sterilized Kanamycin100 ug/ml?).
  13. Transfer some bacteria to another antibiotic-containing plate using a loop.
  14. Incubate plates at 37°C for 18 h.

Visual Protocol

2011-05-11 visual protocol

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