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High Electrotransformation Efficiencies Obtained With DNA From Ligation Mixtures

, 1x ligase buffer (50 mM Tris, pH 7.6, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, and 25 g/ml BSA), and 0.1 Weiss unit/l T4 DNA ligase (Bio-Rad). All ligations reactions were incubated at 16 C overnight. The ligase was heattreated by placing the mixtures at 65 C for 15 minutes.

Dilutions were prepared by removing 1 l aliquots from the ligation mixture and diluting them 1/10 or 1/100 in water. Both ethanol and isopropanol precipitations were carried out by combining 10 ng samples from the ligation reactions with a 0.1 volume of 3 M sodium acetate, 20 g of glycogen (except where noted), and 2.5 volumes of ethanol or 1 volume of isopropanol. The samples were placed on ice for 15 minutes and then pelleted at 14,000 x g for 10 minutes. The pellets were washed with 70% ethanol, dried, and resuspended in TE buffer. cDNA was prepared from the 2.3 kb mRNA supplied with the Superscript plasmid system, according to instructions from the manufacturer (LTI). The cDNA was constructed with SalI and NotI adaptors. The cDNA was size-fractionated by column chromatography to remove residual adaptors and to isolate cDNA larger than 500 bp. A total of 10 ng of cDNA was ligated to 50 ng of pSPORT 1 vector (LTI) using the ligation conditions described above. (The 4.1 kb pSPORT 1 vector contains SalI and NotI restriction sites for directional cloning of the cDNA.)

Electroporation
One l aliquots of the sample DNA were mixed with 40 l of JS5 Electro-Competent E. coli cells. The E. coli Pulser apparatus (Bio-Rad) was set to a voltage of 1.80 kV, and chilled 0.1 cm electrode gap cuvettes were used. Time constants of 4.7 - 4.8 msec were observed. Within 10 seconds of the pulse, 1 ml of SO
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