This is a protocol for transforming E. coli with plasmid DNA.


Category
Protocol
Record History
Added on May 11, 2010 at 4:39 PM by Lindner, Jill
Modified on July 9, 2010 at 10:20 AM by Lindner, Jill
Shared with (contributions)
VCSCBi: Training / Protocols Master

Reagents, supplies, and equipment:

  • E. coli competent bacteria
  • 1.5 ml Eppendorf tubes
  • 42°C water bath
  • LB broth without ampicillin (no Amp)
  • LB + Amp plates
  • 100 mM IPTG
  • 2% X-gal
  • 37°C shaker rack
  • microcentrifuge

Procedure: After ligation of vector and insert is complete, the ligation mixture is ready to be transformed into bacteria.

  1. Add 200 µl E. coli competent bacteria to all of the plasmid ligation mixture in a 1.5 ml Eppendorf tube.  Place on ice for 30 min.
  2. Heat shock at 42°C X 1 min.
  3. Add 1 ml of LB broth (No Amp) to cells.  Incubate 1 hr at 37°C.
  4. Pre-warm 2 LB + Amp plates per transformation at 37°C.
  5. Transfer a 200 µl aliquot of transformation mixture to a clean Eppendorf tube (low dilution).
  6. Microfuge to pellet the cells (Be careful since too much centrifugation will destroy the cells).  Quickly turn microfuge ON and OFF 3-4X.  Pipette off and discard all but 200 µl of solution (high dilution).
  7. If blue/white color selection can be used, to the low and high dilutions, add:

                    10 µl 100 mM IPTG

                    50 µl 2% X-gal

  8. Transfer mixtures to separate, pre-warmed LB + Amp plates. Spread with a bent glass rod.
  9. Incubate 8-12 hr, inverted, at 37°C.
  10. Pick white colonies and suspend in 2.5 ml LB + Amp broth (1 colony per tube).  Incubate on shaker rack at 37°C 8 hr to overnight.
  11. Suspensions are ready for mini-prep assay.