Dorman:Lambda Red mediated Recombination

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (Aded link to tetRA amplification)
(Small note about what to do if no transformants)
Line 31: Line 31:
- 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 ad 1 mL of L, mix, and pour the entire contents of the cuvette into a 15 mL centrifuge tube.
+
- 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.
Line 37: Line 37:
- 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 11:54, 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

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