Dorman:Lambda Red mediated Recombination

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- Take the flasks out of incubator and leave them on an ice/water slurry until the flask is cold.  (This took me ~5 min.  Remember, an ice/water slurry chills flasks much faster than just ice because the water is a better conductor of heat than air.)
- Take the flasks out of incubator and leave them on an ice/water slurry until the flask is cold.  (This took me ~5 min.  Remember, an ice/water slurry chills flasks much faster than just ice because the water is a better conductor of heat than air.)
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- Transfer the cells into a 50 mL tube, and spin at 4000 rpm in the bucket centrifuge for 7 minutes.  (You can centrifuge for longer, but this will result in a more dense pellet that takes more agitation to resuspend in the next step.
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- Transfer the cells into a 50 mL tube, and spin at 4000 rpm in the bucket centrifuge for 5 minutes.  (You can centrifuge for longer, but this will result in a more dense pellet that takes more agitation to resuspend in the next step.
- Pour off the supernatant, add 25 mL of water, and re-suspend by swirling.  (You shouldn't need to pipette, and certainly don't vortex!)
- Pour off the supernatant, add 25 mL of water, and re-suspend by swirling.  (You shouldn't need to pipette, and certainly don't vortex!)

Revision as of 09:09, 10 July 2008

We've now done λ-Red-Mediated recombination with E. coli K-12, Shigella flexneri BS184 and Salmonella Typhimurium SL1344. The protocol below is mostly from Karlinsey [1].

If you're doing you knockouts with the tetRA element, we've got a protocol for that.

Day 0:

- Streak out your strain w/ pKD46 (you can get this plasmid from Dan) onto L/Carb and grow o/n at 30°C.

Day 1:

- Pick a single colony off the plate into 1 mL of L in a tube, vortex, and then add the entire volume of this to 25 mL L + 100 μg/mL carbenicillin in a 250 mL flask.

- Grow at 30°C utill the culture reaches an OD600 of between 0.4 to 0.6.

- Add L-arabinose to a final concentration of 0.2% (e.g. 250 μL of 20% arabinose)

- Grow for 1 hour.

- While the cells are inducing, turn on the bucket centrifuge so that it will be at 4°C. Also leave 50 mL of sterile water on ice.

- Take the flasks out of incubator and leave them on an ice/water slurry until the flask is cold. (This took me ~5 min. Remember, an ice/water slurry chills flasks much faster than just ice because the water is a better conductor of heat than air.)

- Transfer the cells into a 50 mL tube, and spin at 4000 rpm in the bucket centrifuge for 5 minutes. (You can centrifuge for longer, but this will result in a more dense pellet that takes more agitation to resuspend in the next step.

- Pour off the supernatant, add 25 mL of water, and re-suspend by swirling. (You shouldn't need to pipette, and certainly don't vortex!)

- Pellet the cells and repeat the same wash.

- Resuspend the cells in 250 μL of water. They are now ready for electroporation.

- Add 50 μL of cells and 100 to 200 ng of DNA into a 2mm cuvette. Eletroporate at 200Ω, 2.5 kV, and 25 μF.

- Immediately add 1 mL of L, mix, and pour the entire contents of the cuvette into a 15 mL centrifuge tube.

- Grow for 1 hr with shaking at 37°C.

- Plate 500μL of cells on the appropriate selective media. Leave the rest of the culture to grow o/n in the shaking incubator in case you get no transformants.

Day 2:

-Hopefully you've got transformants. Streak these again on a plate with the appropriate selective media to purify the mutant. If you have no transformants, plate the remaining amount of cells.

N.B. I strong reccomend that once you've made your mutation, you transduce it into a fresh genetic background with phage P1 (for E. coli or Shigella) or P22 (for Salmonella). This is because the λ-Red proteins, in addition to recombining your element, may have recombined regions of the chromosome (like IS elements) resulting in deletions. This would result in your strain having not only the mutation that you want, but another that you don't even know about!

References

  1. Karlinsey JE. . pmid:17352924. PubMed HubMed [Karlinsey-2007]
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