Transformation of E. coli with plasmid or ligation reaction.
Techniques have been developed to allow bacterial and eukaryotic cells to absorb DNA
molecules which may then replicate in the cells. The process is referred to as transformation.
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Transformation of chemically-competent cells usually results in ~10 transformants per ug
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DNA. Electro-competent cells give higher transformation efficiencies, around ~10
transformants per ug DNA.
Chemically-competent Transformation
1.- Gently thaw frozen competent bacterial cells on ice. Aliquot 50 ul chemically-competent
E. coli cells (e.g. strain JM109 or DH5α) into an appropriate number of sterile, labeled 1.5 ml
tubes on ice (keeping cells ice cold at all times is required for good transformation
efficiency). Sterile 6-10 ml falcon tubes can also be used and may give higher efficiencies.
2.- Add 2-5 ul DNA to cells, mix gently by flicking tube. Both a positive and a negative
control are required. Use 1 ng of plasmid for positive and no DNA as negative control.
3.- Incubate cells/DNA on ice for 10 min.
4.- Heat shock at 42 C in water bath for exactly 50 seconds. Return immediately to ice for 2
minutes.
5.- Add 0.950 ml LB or SOC media to the cells and incubate at 37 C at 200 rpm for 30 min
to 60 minutes (use a Styrofoam tube holder placed in flask holder as a holder for the 1.5 ml
tubes). Kan selection requires at least 1 hour of recovery time.
6.- Plate 100 ul of cells on LB plates containing appropriate antibiotics. After plates have
dried completely, incubate overnight at 37 C. Save leftover cells in refrig. for 1-2 days, just
in case. You can also plate the remaining cells by spinning down cells for 1 minute, pouring
off most of the media and resuspending the cells in remaining media and plating onto another
plate.
Electro-competent Transformation
1.- Thaw electro-competent E. coli cells on ice, flick to mix and keep on ice. Aliquot 40 ul
thawed cells into an appropriate number of sterile, pre-chilled, labeled 1.5 ml tubes, taking
into account a negative control that lacks DNA, on ice. A positive transformation control
should also be done – which can also show you how good (how efficient) your cells are. Use
1 ul of a plasmid (diluted to 0.1 ng/ul).
2.- Add 1-3 ul of DNA to cells, mix gently by flicking tube and return to ice.
3.- Incubate on ice for 10 min. Place appropriate number of electro-cuvettes on ice to chill.
4.- Transfer cells/DNA mix to well of cuvettes; tap to mix. Save 1.5 ml tubes for next step.
Shock cells with Bio-Rad electroporator set on EC1 (for E. coli, 1 mm cuvette, which is the
size we have in our lab). Immediately after shocking a cuvette, go to step 5 (i.e., don’t shock
all before going to step 5). If the electroporator shows “arc” after shocking your cells, you
must repeat the transformation using less DNA. Alternatively, this might mean your cells
were not sufficiently washed if they are homemade.
5.- After shocking a cell/DNA solution, add 0. 950 ml LB or SOC liquid media to the cells,
very gently pipet cells up/down and transfer cells back into original 1.5 ml tube.
6.- Incubate cells at 37 C at 200 rpm for 30 min to 60 minutes. (use a Styrofoam tube holder
placed in a flask holder in the incubator in Cell lab as a holder for the 1.5 ml tubes). For kan
selection, make sure you incubate cells for at least 60 minutes.
7.- Plate 150 ul of cells on LB plates containing appropriate antibiotics. After plates have
dried completely, incubate overnight at 37 C. Save leftover cells in refrigerator. for 1-2 days,
just in case you don’t get any colonies.