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 waste are we gonna generate
- hot water; combusted propane gas
- Thaw competent cells (Lyocomp E. coli) on ice.
- During this time pre-chill sterile centrifuge tubes on ice.
- Gently tap competent cell containers to mix
- Aliquot bacterial suspension in pre-chilled tubes (100 µl per tube).
- To the aliquots add 1 µl of plasmid (RFP PUC19).
- Gently tap centrifuge tubes to mix
- Incubate 30 min on ice.
- During this time pre-heat water bath to 42°C (don’t use heating block).
- Incubate samples at 42°C in the water bath for 60s. Do not shake.
- Immediately chill samples on ice for 2 minutes.
- Add 0.95 ml of room temperature SOC.
- Plate 10 µl of bacterial suspension on antibiotic-containing agar plate. (filter sterilized Kanamycin100 ug/ml?).
- Transfer some bacteria to another antibiotic-containing plate using a loop.
- Incubate plates at 37°C for 18 h.
<a href="http://www.flickr.com/photos/macowell/5713410863/" title="2011-05-11 visual protocol by macowell, on Flickr"><img src="http://farm4.static.flickr.com/3421/5713410863_fc62439a0e.jpg" width="500" height="374" alt="2011-05-11 visual protocol"></a>