The following is a historic record. Find the latest protocol here.
- at least 20 mL of LB media
- two 14 mL culture tubes
- two sterile microcentrifuge tubes AKA Eppendorf tubes (1.7 mL capacity each)
- overnight liquid cultures of Agar Art E. coli, at least 1 mL each:
- Pink
- GFP
- 1000X chloramphenicol stock solution (in BosLab −20°C freezer), at least 20 µL
- Three micropipettors of the following types, plus compatible tips:
- at least 10 mL 1M NaCl sterile solution
- 1M means 1 molar concentration, i.e. 1 mole NaCl per liter of solution
- at least 50 mL 1 M trehalose sterile solution
- Dry culture carrier comprising either:
- sterile petri dishes and pieces of filter paper cut to fit in them, or
- new unused zip lock bags and pieces of filter paper cut to fit in them
- 37°C plate incubator to use for drying cells (consider drying at 30°C if cells don't revive)
- 37°C shaker incubator for liquid cultures
- tube vortexer
- PCR workstation, or laminar flow workbench, or clean work surface with bunsen burner to create a sterile airspace in which to work
- Work in a sterile airspace such as the BosLab PCR workstation (a.k.a. “PCR hood”).
- I labeled 2 culture tubes with my initials, the date, and as follows:
- LB+chloro GFP
- LB+chloro Pink
- With a P1000 micropipette, transfer 900 µL LB media to each tube, TWICE, for a total of 1800µL LB into each tube.
- Using a fresh P1000 tip, transfer 1000 µL of 1M (1 molar) NaCl to each tube THREE TIMES, for a total of 3000 µL into each tube.
- With a P20 micropipette, transfer 5 µL of 1000X chloramphenicol stock solution to each of the “LB+chloro” tubes.
- Vortex each tube for 10 seconds so contents are well mixed.
- Using a P200 micropipette to transfer 195 µL GFP Agar Art E. coli liquid culture to the LB+chloro GFP tube.
- Same with the Pink culture into the LB+chloro Pink tube.
- Incubate tubes in the orbital shaker at 37°C and 260 RPM for 36 hours. See Ref. 5 below for justification. Alas, we were sharing the BosLab incubator with a long-running 30°C project, so we incubated at that temperature. Also, because of scheduling issues, we ended up incubating for 67 hours.
- While incubating, label two sterile microfuge tubes:
- LB+chloro Pink
- LB+chloro GFP
- Label 3 sterile Petri dishes: LB+chloro Pink
- Label 3 sterile Petri dishes: LB+chloro GFP
- Sterile transfer a piece of autoclaved paper into each Petri dish.
- Pipette 1500 µL from each incubated culture tube to the correspondingly labeled microfuge tube.
- We used a P1000 and pipetted 750 µL twice.
- Centrifuge the microfuge tubes to pellet cells. Discard supernatant into liquid biohazard waste.
- Resuspend cells in 150 µL of 1 M trehalose by vortexing for 15-30 seconds, or until the pellet is gone.
- Pipette 50 µL per piece of paper. This covers an area of approximately 1 cm x 1 cm.
- Next time, laser-print some boxes on the paper so we know where to pipette the culture.
- Put paper in sterile petri dishes with lids closed but NOT parafilmed or sealed. Use two small pieces of tape to keep the lids from opening if the dishes are dropped.
- Or put paper in new unused zip lock bags (leave bags open). This might work also but we haven't tried it yet.
- Place petri dishes or bags in plate incubator to dry. May take 1-3 days depending on ambient humidity.
- Due to scheduling, we incubated for a full 7-day week.
- Remove petri dishes and/or bags and store in a cool dry place.
TBD: rehydration procedure using LB. Consider rehydrating both in broth and in agar.