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Abstract
The plasmid p328.5 was used to transform competent Escherichia coli cells, using either the Hanahan or electroporation protocols, and the resulting transformation efficiencies were compared. In each case, competent cells were transformed under four different conditions: (i) plasmid DNA alone, or the plasmid in addition to chromosomal DNA from either: (ii) E. coli HB101, (iii) E. coli B23 or (iv) Bacillus subtilis WB746. Results from the Hanahan protocol showed that, as expected, there was a reduction in transformation efficiency when cells were transformed by both the plasmid and chromosomal DNA, with DNA from B. subtilis having the largest net decreasing effect. Upon comparison with the results of electroporation transformation, the opposite was seen; cells transformed with both plasmid and chromosomal DNA from B. subtilis showed the highest net rate of transformation efficiency. These results indicated that host cell or foreign chromosomal DNA does reduce transformation efficiencies in the Hanahan protocol. Few conclusions can be drawn from the electroporation results, which appeared erratic and random. These results may have been attributable to differences in the lengths of the chromosomal DNA fragments used. Competent E. coli cells, for both the Hanahan and electroporation methods, were frozen in either CaCl (chemically competent cells), sterile distilled water (electrically competent cells) 2 or a glycerol cryoprotectant solution and their viability was tested after 48 hours at –20°C. Our results showed that most of the cells died despite the presence of glycerol when frozen at –20°C. Some growth of competent cells frozen with glycerol was seen on nutrient agar plates, but the number of colonies on a final count was still low after more than 48 hours growth. No colonies were seen from samples frozen without any glycerol.