Overview
This a protocol for preparing and handling E. coli cells for electroporation. This protocol has mostly been adapted from the Noyes Lab protocol. A simpler protocol from NEB does exist though it did not work for me initially. I have, however, used a similar protocol in the past with good results, so it’s linked to in the ‘Notes’ section.
Materials
- 1 L flask
- 1 L 2xYT
- Timer
- 1000x tetracycline
- USOΩ starter culture
- 50x 1.5 mL micro-centrifuge tubes
- 4x 250 mL conical bottom flasks
- 500 mL 10% glycerol
- 1 L sterile water
- Large ice bucket (I like to use the big autoclave trays)
- 5x 10 mL pipettes
- 1 mL & 200 uL filter tips
- Spectrophotometer cuvette
- Dry ice/liquid nitrogen bucket
Making USOΩ Starter Cultures
Cells from the original USOΩ glycerol stock bMM370 (E. coli, ΔrpoZ ΔpryF ΔhisB) were streaked out on 2xYT media (Becton Dickinson, 244020) bacto agar (Becton Dickinson, 214030) containing 10μg/mL Tetracycline hydrochloride (Fisher, BP912-100) and 50μg/mL Zeocin (Invitrogen, 46-0509) and allowed to grow overnight. A single colony was picked the next day and was used to inoculate sterile 2xYT media containing 10μg/mL Tetracycline hydrochloride. This was allowed to grow overnight (no longer than 16 hrs) on a roller drum at 37°C. The next morning, 1mL of the saturated culture was used to inoculate 1L of sterile 2xyt media with 10μg/mL Tetracycline hydrochloride. Shaking at 37°C the 1L culture was allowed to expand to an OD of .6. It was then moved into 4, 250mL conical bottom flasks (Corning, 430776) and spun down using rotor J5.3 at 5,000 rpm (~4,000 xg) in a Beckman Coulter Avanti J-E centrifuge for 15 minutes at 4°C. The supernatant was decanted and the pellets were re-suspended in a total of 30 mL of 2xyt media plus 20% (w/v) glycerol (Fisher, BP229-4). Finally, 1mL at a time was transferred to micro-centrifuge tubes and flash frozen on dry ice. These “starter cultures” were sorted at -80°C for later use.
Strain Growth and Harvest
- Measure out 1 L of sterile 2xYT media into a 2 L flask. Add 1 mL of 1000x Tet to flask and swirl to mix.
- Remove 1 USOΩ starter culture from the -80° freezer and allow to thaw on bench.
- Add USOΩ starter culture (approx. 1 mL) to 2 L flask.
- Put flask in shaking incubator at 37°.
- Move sterile water, 10% glycerol, and 4x 250 mL conical bottom flasks to 4° fridge or cold room.
- Culture should take about 3hrs 15min to grow to the appropriate OD (~0.60) [NB: It’s taken up to 4.5 hrs sometimes for me]. At 3hrs, pre-chill the centrifuge to 1° and prepare your cart for the washing procedure.
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- Fill the large ice bucket with ice.
- Fill dry ice bucket (or liquid nitrogen closer to aliquoting in microfuge tubes.)
- Label 50x 1.5 mL micro-centrifuge tubes with date and place in cold room.
- Move 10 mL pipettes and 1 mL & 200 uL tips cold room.
- Remember your timer.
- At 3hrs 15min, measure OD600 (first blank with fresh 2xYT). Aim for measurement of 0.57-0.64 with the ideal being 0.60. If the culture has not grown enough, incubate shaking for longer (after OD 0.40, ~15min = ~0.08 OD).
- Once the culture reaches the desired density, remove from shaker and rapidly cool culture flask in large ice bucket in cold room.
- AT THIS POINT ALL LIQUID HANDLING STEPS SHOULD BE DONE IN COLD ROOM AND BOTTLE TRANSFERS TO/FROM CENTRIFUGE SHOULD BE ON ICE
- Pre-chill centrifuge to 4°.
- Once entire culture is cooled to approximately 4°, pour, balanced, into four conical bottom flasks.
- Spin conical bottom flasks at 5000 rpm for 10 min.
- Decant supernatant and pellet in ~20 mL sterile water. Combine 4 pellets into 2 bottles.
- Fill flasks with sterile water up to 250 mL. Spin at 4000 rpm for 10 min.
- Repeat wash with sterile water. Resuspend 2 pellets in 225 mL sterile water. Repeat spin.
- Decant supernatant, and wash with 150 mL 10% glycerol per conical. Spin at 4000 rpm for 10 min.
- Decant supernatant, resuspend pellet in ~20 mL 10% glycerol, and combine 2 pellets into one flask. Fill with 10% glycerol to 150 mL.
- Spin at 4000 rpm for 13 min.
- Completely decant supernatant (some minor pellet loss is expected). Resuspend pellet in 2.75 mL 10% glycerol.
- In cold room, aliquot 88 uL cell suspension into each labeled micro-centrifuge tube and flash freeze in dry ice-ethanol bath or liquid nitrogen. Keep flask on ice in cold room.
- Once cell suspensions are frozen, move tubes to -80° freezer.