McClean:E. coli Electroporation

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.

Stock Solutions

Stock Solution 1

• This is a very simple solution, so we only need a one line description of how to make it.

Stock Solution 2

This is a more involved solution, so we will describe how to make it in several steps:

1. Step 1
2. Step 2
3. Step 3

Protocol

1. 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.
2. Remove 1 USOΩ starter culture from the -80° freezer and allow to thaw on bench.
3. Add USOΩ starter culture (approx. 1 mL) to 2 L flask.
4. Put flask in shaking incubator at 37°.
5. Move sterile water, 10% glycerol, and 4x 250 mL conical bottom flasks to 4° fridge.
6. 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. Add enough water to allow ice to swirl.
  • Fill dry ice bucket and place on cart.
  • Label 50x 1.5 mL micro-centrifuge tubes with date and place on cart.
  • Move 10 mL pipettes and 1 mL & 200 uL tips to cart.
  • Remember your timer.

1. At 3hrs 15min, measure OD using the Nanodrop (first blank with fresh 2xYT). Aim for a 600 nm 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).
2. Once the culture reaches the desired density, remove from shaker and rapidly cool culture flask by swirling in large ice bucket.
3. Once entire culture is cooled to approximately 4°, pour, balanced, into four conical bottom flasks.
4. Remove sterile water and 10% glycerol from the fridge and place in ice bucket on the cart. Take entire cart to pre-chilled centrifuge.
5. Spin conical bottom flasks at 5000 rpm for 10 min.
6. Decant supernatant and pellet in ~20 mL sterile water. Combine 4 pellets into 2 flasks.
7. Fill flasks with sterile water up to 250 mL. Spin at 4000 rpm for 10 min.
8. Repeat wash with sterile water. Resuspend 2 pellets in 225 mL sterile water. Repeat spin.
9. Decant supernatant, and wash with 150 mL 10% glycerol per conical. Spin at 4000 rpm for 10 min.
10. Decant supernatant, resuspend pellet in ~20 mL 10% glycerol, and combine 2 pellets into one flask. Fill with 10% glycerol to 150 mL.
11. Spin at 4000 rpm for 13 min.
12. Place dry ice around micro-centrifuge tubes to chill them.
13. Completely decant supernatant (some minor pellet loss is expected). Resuspend pellet in 2.75 mL 10% glycerol.
14. Aliquot 88 uL cell suspension into each labeled micro-centrifuge tube and flash freeze on dry ice. Keep flask on ice.
15. 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.