McClean:E. coli Electroporation: Difference between revisions

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Line 23: Line 23:
#Add USOΩ starter culture (approx. 1 mL) to 2 L flask.
#Add USOΩ starter culture (approx. 1 mL) to 2 L flask.
#Put flask in shaking incubator at 37°.
#Put flask in shaking incubator at 37°.
#Move sterile water, 10% glycerol, and 4x 250 mL conical bottom flasks to 4° fridge.
#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.
#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 the large ice bucket with ice.  
**Fill dry ice bucket and place on cart.
**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 on cart.
**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 to cart.
**Move 10 mL pipettes and 1 mL & 200 uL tips cold room.
**Remember your timer.
**Remember your timer.
#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).
#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 by swirling in large ice bucket.
#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.
#Once entire culture is cooled to approximately 4°, pour, balanced, into four conical bottom flasks.
#Remove sterile water and 10% glycerol from the fridge and place in ice bucket on the cart. Take entire cart to pre-chilled centrifuge.
#Spin conical bottom flasks at 5000 rpm for 10 min.
#Spin conical bottom flasks at 5000 rpm for 10 min.
#Decant supernatant and pellet in ~20 mL sterile water. Combine 4 pellets into 2 flasks.
#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.
#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.
#Repeat wash with sterile water. Resuspend 2 pellets in 225 mL sterile water. Repeat spin.
Line 41: Line 42:
#Decant supernatant, resuspend pellet in ~20 mL 10% glycerol, and combine 2 pellets into one flask. Fill with 10% glycerol to 150 mL.
#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.
#Spin at 4000 rpm for 13 min.
#Place dry ice around micro-centrifuge tubes to chill them.
#Completely decant supernatant (some minor pellet loss is expected). Resuspend pellet in 2.75 mL 10% glycerol.
#Completely decant supernatant (some minor pellet loss is expected). Resuspend pellet in 2.75 mL 10% glycerol.
#Aliquot 88 uL cell suspension into each labeled micro-centrifuge tube and flash freeze on dry ice. Keep flask on ice.
#In cold room, aliquot 88 uL cell suspension into each labeled micro-centrifuge tube and flash freeze on dry ice or liquid nitrogen. Keep flask on ice in cold room.
#Once cell suspensions are frozen, move tubes to -80° freezer.
#Once cell suspensions are frozen, move tubes to -80° freezer.


Revision as of 12:15, 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

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 or cold room.
  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.
    • 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.
  1. 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).
  2. Once the culture reaches the desired density, remove from shaker and rapidly cool culture flask in large ice bucket in cold room.
  3. AT THIS POINT ALL LIQUID HANDLING STEPS SHOULD BE DONE IN COLD ROOM AND BOTTLE TRANSFERS TO/FROM CENTRIFUGE SHOULD BE ON ICE
  4. Pre-chill centrifuge to 4°.
  5. Once entire culture is cooled to approximately 4°, pour, balanced, into four conical bottom flasks.
  6. Spin conical bottom flasks at 5000 rpm for 10 min.
  7. Decant supernatant and pellet in ~20 mL sterile water. Combine 4 pellets into 2 bottles.
  8. Fill flasks with sterile water up to 250 mL. Spin at 4000 rpm for 10 min.
  9. Repeat wash with sterile water. Resuspend 2 pellets in 225 mL sterile water. Repeat spin.
  10. Decant supernatant, and wash with 150 mL 10% glycerol per conical. Spin at 4000 rpm for 10 min.
  11. Decant supernatant, resuspend pellet in ~20 mL 10% glycerol, and combine 2 pellets into one flask. Fill with 10% glycerol to 150 mL.
  12. Spin at 4000 rpm for 13 min.
  13. Completely decant supernatant (some minor pellet loss is expected). Resuspend pellet in 2.75 mL 10% glycerol.
  14. In cold room, aliquot 88 uL cell suspension into each labeled micro-centrifuge tube and flash freeze on dry ice or liquid nitrogen. Keep flask on ice in cold room.
  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

or instead, discuss this protocol.


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