Change from 2021 Aug 1 revision: Reduce culture volume from 5 mL to 1 mL so each culture can fit in a 5 mL tube. Increase from 4 colors to 6.
Materials and equipment needed for N colors: (N=6 for us)
| For N=6 different colored strains: | |
| 2 × N sterile 5 mL culture tubes with caps | 16 culture tubes = 12 culture tubes + 4 control tubes |
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4 sterile 5 mL culture tubes, with caps, as controls:
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| Rack/holder for above culture tubes | I used 2 racks with 16 open spaces each so I could start with all the tubes on one rack. Then as I perform a protocol step on a tube, I can move it to the other rack to indicate that that tube has completed the current step. This helps me track my progress in case I get distracted while performing the protocol (e.g. a lab partner needs help). |
| Marker pen for labeling tubes | Use a VWR Lab Marker |
| Stock bottle of at least (2N+4+2) x 1 mL of sterile liquid LB media |
Liquid LB amount needed: 2N+4+2 = 2x6 + 4 + 2 = 18 We'll use 20 mL, which is 18 mL plus 2 mL extra just in case |
| 1 sterile serological pipette to aliquot (2N+4+2) x 1 mL | could use a 25 mL sterile disposable pipette to transfer the 20 mL aliquot (should we use a Falcon tube to receive the aliquot?) |
| pipettor for serological pipettes (e.g. pipette-aid) | will need to check that the pipettor doesn't leak and has good batteries |
| a pipette to transfer 1 mL (i.e. 1000 µL) | will use a P1000 set to 1000 µL |
| 1000X chloramphenicol stock solution, 25 mg/mL, from the −20°C freezer (they are still liquid at −20°C) |
1000x chloroamphenicol stock solution needed: 20 µL (i.e. use 1 µL stock solution per mL of media) |
| micropipettor to deliver N × 10 µL | use a P200 (i.e. 200 µL capacity pipettor) |
| 1 sterile micropipette tip for the above | get the box of P200 tips (I realize they've been opened and aren't guaranteed to be sterile – I think the chance of contamination from a new P200 tip is small – though if the protocol fails we'll try pipetting 70% ethanol first before retrying.) |
| sterile culture media bottle to hold the LB media aliquot mixed with chloramphenicol | an empty, clean, autoclaved 100 mL glass media bottle |
| (2 × N + 4) sterile inoculation loops all from the same bag or container | 16 sterile inoculation loops |
| Plates with streaked, colorful, agar art colonies from the fridge or incubator | the 6 single-color agar plates from the fridge marked “Frank Lee” |
| (Optional) Eppendorf tube of 70% ethanol for sterilizing a P200 tip. | Use a 5 or 10 mL serological pipette to aliquot an inch or so of 70% ethanol into a 1.5 mL Eppendorf tube. Use this as an opportunity to test that your serological pipettor has good batteries and doesn't leak. |
| Laminar flow hood | reserve the Boslab PCR Workstation |
Protocol: (quantities are for N=6 colors)
Parent project: Maintaining Agar Art strains at BosLab.