(r2) ProcedureGenomeModificationDatsenkoWanner < Lab

---++ Lambda Red Protocol
 
---++ Lambda Red Plasmid

pKD46 (ampR)

---++ Making Competent Cells expressing Lambda Red

---+++ Day 1

   * Grow starter culture (of strain of interest with pKD46) in 2mL LB-Amp overnight at 30°C

---+++ Day 2

   * Dilute 1:80, 500 uL -> 40mL SOB-Amp. (LB can also be used, but SOB is preferred)
   * Grow on 30 deg shaker until OD600 = ~ 0.350 - 0.400, should take 2-3 hours
   * Induce cultures with filtered 10% Arabinose creating a final concentration of 0.1% Arabinose  
   * Place back on 30°C shaker for 15 minutes
   * After induction, immediately transfer culture to 50mL falcon tube and place on ice for 40 min
   * Spin cultures 3000rpm x 15 min to pellet cells.  
   * Resuspend pellet in 35 mL cold ddH20 
   * Spin 5000 rpm for 10 minutes 
   * Wash again with 30 mL ddH20 or 10% Glycerol if making frozen stocks Spin 7000rpm for 10 minutes. Dump out water, shake off H20 gently, careful not to disturb pellet. Return tubes to ice bucket.  Allow time for residual liquid in tube to collect on pellet.  Swirl to resuspend pellet.
   * Aliquot 50 uL cell suspension into cold eppendorf tubes.  1 for each electroporation

*NOTE* 40 mL culture should produce approximately 250-350 uL of cells, enough for 5-7 50 uL electroporations.  Dilute cells with ddH20/10% glycerol if needed.

---++ Transforming Cells

   * Add DNA to cell aliquots, typically 50-200ng (be sure to desalt)  Transfer cells + DNA to cold 1mm (or 2mm) gap cuvettes
   * Electroporate, recover immediately with 1mL SOC (draw SOC into pipette tip before electroporating)
   * Transfer to culture tube, place on 37°C shaker for 1.5 hrs  
   * Plate 100-200 uL of recovery onto selection plate. Leave recovery culture on bench O/N, plate more next day if unsuccessful
   * Grow selection plate 37°C overnight; Colonies may a full 24 hours to appear

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

---+++ 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)
Barrick Lab > ProtocolList > ProcedureGenomeModificationDatsenkoWanner
Topic revision: r2 - 2012-06-14 - 21:21:40 - Main.JeffreyBarrick