DIYbio/BOSSlab/Notebook/2011/05/15

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.

Notes

• 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.

Ian's writeup

• 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.

Thanks,
- Ian