Dorman:Lambda Red mediated Recombination: Difference between revisions
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- 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. | - 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 | - 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. | - Grow for 1 hr with shaking at 37°C. | ||
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- 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. | - 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 3:''' Hopefully you've got transformants | '''Day 3:''' 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. | ||
===References=== | ===References=== | ||
Revision as of 08:54, 27 May 2008
We've had trouble getting λ-Red-Mediated recombination to work, but today (21 March 08) I finally got it to work well. The protocol below is mostly from Karlinsey [1]. I'll modify/update this as we learn more.
If you're doing you knockouts with the tetRA element, we've got a protocol for that.
N.B.: All the below was done with E. coli K-12. Please add a note if you find that you need to modify this for Shigella or Salmonella
Day 0: Streak out MG1655 w/ pKD46 (you can get this strain 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 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.
- 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 3: 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.
References
- Karlinsey JE. lambda-Red genetic engineering in Salmonella enterica serovar Typhimurium. Methods Enzymol. 2007;421:199-209. DOI:10.1016/S0076-6879(06)21016-4 |
- Dan Stoebel 12:50, 21 March 2008 (CDT):
