Lambda Red Protocol

Lambda Red Plasmids

  • pKD46 (ampR)
  • pKD78 (camR)

Making Competent Cells expressing Lambda Red

Day 1

  • Grow starter culture (of strain of interest with pKD46/pKD78) in 10mL LB-Amp or LB-Cam overnight at 30°C

Day 2

  • Add 500uL of overnight culture to 30mL LB-Amp (or Cam).
  • Grow on 30C shaker until OD600 = ~ 0.350 - 0.400, (takes ~2-3 hours)
  • Induce cultures with filtered 10% Arabinose, creating a final concentration of 0.1% Arabinose (300uL of 10% Arabinose into the 30mL of culture).
  • Place back on 30°C shaker for 15 minutes, then transfer to 50mL falcon tube, and place on ice for 40 minutes. Start chilling centrifuge to 4C.
  • Spin cultures @ 1800 x g for 15 min to pellet cells.
  • Resuspend pellet in 30 mL cold water.
  • Spin @ 3000 x g for 5 minutes, resuspend in cold water.
  • Spin @ 3000 x g for 5 minutes, and resuspend in cold 10% glycerol.
  • After this spin cycle, while pouring out the glycerol pay close attention to the pellet. It is likely somewhat loose/mushy at this point, so pour off the glycerol until the pellet starts to move, then briefly vortex to resuspend the entire pellet, and transfer the entire remaining volume to a cold 15mL tube. Remaining volume will likely be between 10 to 3 mL.
  • Rebalance centrifuge, spin @ 3000 x g for 5 minutes. Pour off all glycerol, pellet should be stable.
  • Add back cold 10% glycerol for desired cell density, and number of elctroporations. Usually 300uL will yield a good cell density for ~6 electroporations. Resuspend pellet by pipetting with 1mL pipette.
  • Aliquot 50 uL cell suspensions into cold eppendorf tubes.

Transforming Cells

  • Add DNA to cell aliquots, typically 50-200ng (be sure to desalt, or use very low volumes of DNA i.e., 1uL)
  • Transfer cells + DNA to a chilled gap cuvette
  • Electroporate, recover immediately with 1mL SOC/SOB/LB.
  • Transfer to a culture tube with 10mL SOC/SOB/LB, place on 30°C shaker for 1.5 hrs.
  • Plate 100-200 uL of recovery onto selection plate (such as LB-Kan if using the Kan cassette from the keio collection). Leave the recovery culture on bench overnight, plate more next day if the initial plating was unsuccessful.
  • Grow the selection plate at 37°C overnight; Colonies may take a full 24 hours to appear.

NOTE Recover at 30 deg if you intend on maintaining pKD46. Plasmid is usually lost at 37°C

Expected Results

The data below used 20ng of purified PCR product (900bp) targeting the kan cassette from the topB keio strain, and 50uL of lambda-ready cells. 3 electroporations per strain.

REL606

Colonies on selective plate Colonies on control plate Dilution Factor Recombination Efficiency
5 65 10^5 7.7E-8  
8 37 10^5 2.2E-7  
7 53 10^5 1.3E-7  

BW25113

Colonies on selective plate Colonies on control plate Dilution Factor Recombination Efficiency
1 135 10^5 7.4E-9  
1 152 10^5 6.6E-9  
3 125 10^5 2.4E-8  

References

  1. Datsenko, K.A. & Wanner, B.L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640-6645.

Contributors

  • Originally from Erik Quandt (6/7/2011)
  • Edited by Steven Sowa (6/2011)
  • Edited by -- Main.NeilGottel - 04 Sep 2012
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Contributors to this topic Edit topic JeffreyBarrick, NeilGottel, DanielDeatherage, SteveSowa
Topic revision: r4 - 2012-09-04 - 17:35:46 - Main.NeilGottel
 
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