DIYbio/BOSSlab/Notebook/2011/05/15

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Photos: http://www.flickr.com/photos/macowell/sets/72157626730414854/

Preflight
what's our goal: transformation of Invivogen lyocomp cells w/ BBa_J04450 on card (circa 2008).

Materials

 * lyocomp E. coli 116, (k12 derived?), non-pathogenic. http://www.invivogen.com/family.php?ID=189
 * BBa_J04450 plasmid-on-card. Ampicillin resistant, based on pSB1A4. http://partsregistry.org/Part:pSB4A1
 * 1000x stock of ampicillin
 * LB-agar (1 L)
 * SOC, 1.8 mL
 * pressure sterilizer, burner, propane
 * ice, water, 50g scale, fridge, freezer, incubator, p200, p1000, tips, gloves, bleach

what are the risks to us

 * fire hazard: pressure sterilizer + propane burner
 * chemical hazard: bleach
 * chemical hazard: concentrated ampicillin
 * experimental hazard: lab-grade E. coli contamination on bench surfaces
 * temperature hazard: molten LB-agar

what waste are we gonna generate

 * bleach + LB-agar (solid)
 * combusted propane gas
 * water
 * tips used to transfer & mix transformed E. coli (bleached)

mitigation of community risks

 * chemical hazards: propane leak in air
 * only use burner outdoors on concrete
 * e. coli on tips
 * dispose of pipet tips into bleach solution, bag & throw away.

Protocol
based on http://www.invivogen.com/PDF/LyoComp_GT116_TDS.pdf


 * 1) Thaw competent cells (Lyocomp E. coli 116) & rehydration solution on ice.
 * 2) Chill 1.5 mL centrifuge tubes on ice.
 * 3) Rehydrate lyophilized cells with 0.5 mL "rehydration solution"
 * 4) Let cells rehydrate for 30 min on ice
 * 5) Add "DNA chads" from biobrick card to 1.5 mL centrifuge tubes
 * 6) dispense 250 mL of rehydrated lyocomp cells to each 1.5 mL centrifuge tube.  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 exactly 30s.
 * 10) Immediately chill samples on ice for 2 minutes.
 * 11) Add 900 uL of room temperature SOC to each tube.
 * 12) Incubate at 37 C for 90 min, shaking at 250 RPM
 * 13) Spread 100 uL of transformed bacteria on LB-Amp plates.
 * 14) Incubate plates at 37°C for 18 h.

Ian's writeup
Thanks, - Ian
 * E. coli transformation with red fluorescent protein.
 * Basic cleanliness techniques - all materials used were edible. We did not eat them :)
 * Competent cells, ampicillin, hot plate, shakers, test tubes, autoclave tape, glassware, pressure sterilizer, pipets, Pitri dishes obtained from industry sources and donations.
 * Concentration of ampicillin unknown, suspect pre-measured x1000.
 * Oven (incubator), refrigerators, meters, bucket, neoprene gloves, bleach (sterilizer) and scales from off-the-shelf and donation sources.
 * RFP plasmid obtained from old diybio kit.
 * Stepped through protocol thrice before doing real transformation.
 * Several times let cells to incubate on ice longer than protocol called for.
 * Used pressure sterilizer and propane frier to sterilize agar, glassware.
 * Temperature regulation of oven/ incubator a issue - try to keep as close to 37 C w/ o going over. Sometimes as cold as 28 C.
 * Made plenty of extra amp lb plates.


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