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IN VIVO SYSTEMS > BUGS > Transformation

> Transformation of competent E coli

> Efficiency

> 5 Minute Method

 

Transformation of Competent E. Coli

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If transforming with supercoil plasmid DNA use as little as possible, the transformation efficiency of most frozen competent cells that we use is in the order of 106 colonies/ug supercoil DNA. (Nicked circular DNA and ssDNA are 50% as efficient, linear DNA is 0.1% as efficient).

When transforming after a ligation reaction there may be some inhibition of DNA uptake because of the pH of the ligation reagents. You can transform up to 2.5 ul of a ligation reaction. If you don't get enough colonies you will have to lower the pH by adding MES buffer or ethanol precipitate the DNA before transforming. If you use MES buffer at 0.1M final, you can transform the entire ligation reaction.

  1. Place 2.5 ul of ligation reaction (or less than 0.5 ul of supercoiled DNA) into a sterile microfuge tube.
  2. Add 50 ul ready-to-use competent E. coli. (Thaw frozen stocks on ice. Do not heat). Place on ice 30 min.  For plasmid DNA I usually suck up 1 ul of DNA into a 100 ul pipet-tip and dispense it back into the tube. Then use the tip to suck up and down the competent cells - there is plenty of DNA stuck to the tip to transform them.
  3. Heat shock tubes at 42oC for 45 sec, add 500ul (up to 1ml) warm (room temperature) LB and incubate at 37ºC for 45 min. This allows the bacteria to express the antibiotic resistance gene in your plasmid (eg. make some beta-lactamase and become AMP resistant). If you leave the bugs at 37ºC longer than 45 minutes they will begin dividing (once every 20 min) and you will gets lots of colonies that came from the same transformant - not what you want!!! 
  4. Spin down bacteria in the microfuge, 5 sec, remove the supernatant resuspend the pellet in 50-100ul antibiotic broth. Plate out the bug suspension on the appropriate agar plate. If using intact supercoil plasmid DNA there is no need to spin down the bugs, just add antibiotic and plate out 50-00ul after incubation.
  5. Incubate the plates at 37oC 12-16 h.

NOTE 1: Most protocols do not suggest the final addition of antibiotic that we use. That is because standard agar plates are much thicker than we use. Our thin plates are much cheaper and less environmentally wasteful. However, since they have less antibiotic surrounding the colony you tend to get satellites unless you add antibiotic when plating out the cells.

NOTE 2: Kanamycin resistant plasmids do not transform well into DH10B or DH5alpha cells; use HB101 or TOPP2 strains instead.