DIYbio/BOSSlab/Notebook/2011/05/11

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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



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