McClean:E. coli Electroporation: Difference between revisions
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#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). | #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. | #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 | #'''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°. | #Pre-chill centrifuge to 4°. | ||
#Once entire culture is cooled to approximately 4°, pour, balanced, into four conical bottom flasks. | #Once entire culture is cooled to approximately 4°, pour, balanced, into four conical bottom flasks. |
Revision as of 12:21, 17 December 2014
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
Protocol
- 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.
- 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.
Notes
NEB electrocompetant cells protocol: https://www.neb.com/protocols/2012/06/21/making-your-own-electrocompetent-cells
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.
Contact
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.