what's our goal:
transformation of Invivogen lyocomp cells w/ BBa_J04450 on card (circa 2008).
- 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
- 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.
based on http://www.invivogen.com/PDF/LyoComp_GT116_TDS.pdf
- Thaw competent cells (Lyocomp E. coli 116) & rehydration solution on ice.
- Chill 1.5 mL centrifuge tubes on ice.
- Rehydrate lyophilized cells with 0.5 mL "rehydration solution"
- Let cells rehydrate for 30 min on ice
- Add "DNA chads" from biobrick card to 1.5 mL centrifuge tubes
- dispense 250 mL of rehydrated lyocomp cells to each 1.5 mL centrifuge tube. 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 exactly 30s.
- Immediately chill samples on ice for 2 minutes.
- Add 900 uL of room temperature SOC to each tube.
- Incubate at 37 C for 90 min, shaking at 250 RPM
- Spread 100 uL of transformed bacteria on LB-Amp plates.
- Incubate plates at 37°C for 18 h.
- p1000 tips don't fit in lyocomp vial. Used p200 tips. Only removed 400 uL (200 uL to each 1.5 mL tube) of rehydrated e. coli from the vial.
- unsure of concentration and age of frozen Amp stock. Assuming 1000x. Poured two LB plates without the amp. Added 100 uL transformant to each.
- distributed 100 uL transformant to two LB-Amp plates.
- incubating one 1.5 mL tube with remaining transformant (900 uL) at 37 C. no shaking.
- storing the other 1.5 mL tube with remaining transformant (900 uL) at 4 C.
- incubation started at 10:57 PM 5/15/2011
- the incubator is very erratic. I'm trying to keep the temperature at or below 37 C. It does not have a very sensitive control loop. I think it is designed to stay +/- 10 C its set point. It's really more of an oven.
- 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.