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.
Place 2.5 ul of ligation reaction (or less than 0.5 ul of supercoiled
DNA) into a sterile microfuge tube.
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.
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!!!
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.
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.