Electrocompetent strains of E. coli (commercially
available or prepared by user) can be transformed by ligation products
prepared with ElectroLigase. Chemically competent cells are also compatible, but
for maximum performance with chemically competent cells, please consider using
the Blunt/TA Ligase Master Mix (NEB #M0367). The
following protocol is recommended by NEB. Other protocols can be used but the
volume of ligation reaction used should not exceed 5 μl reaction per 50 μl
- Thaw competent cells on
- Aliquot 40 μl of cells into a 1.5 ml
microcentrifuge tube on ice.
- Add 2 μl of the ligation reaction to the
cells and mix by finger-flicking. Do not vortex the tube.
- Transfer DNA/competent cell mixture to a
prechilled electroporation cuvette and follow the manufacturers recommendations
for electroporation (e.g. 2500 V, 200 Ω, 25 μF, 2 mm gap
- Add 760 μl recovery media (e.g. SOC) to
the cuvette, mix, transfer the transformed cells to a culture tube and incubate
for one hour at 37°C with shaking (200–250 rpm).
- Spread 50 μl of the outgrowth (undiluted
or diluted 1:5 with recovery media) onto appropriate antibiotic selection plates
and incubate overnight at 37°C.
Transformation efficiencies around 3 x 106
cfu/μg are typically achieved for recombinant blunt-end vectors (vector +
insert), using cells with a 5 x 109 calculated efficiency with uncut
DNA. Results for TA cloning and standard cohesive end (4 bp overhang) cloning
produce even higher numbers, often over 107 cfu/μg. This corresponds
to several hundred colonies on a plate when 50 μl of the outgrowth is plated at
a 1:5 dilution. As with all ligation and transformation protocols, many factors
affect the calculated transformation efficiency, including purity and integrity
of DNA ends, competence of the cells being transformed, media choices,
incubation temperatures and times and biological effects (intact ORF in
high-copy vector, toxic genes, etc.).