1653
Nucleic Acids Research, Vol. 18, No. 6
High-efficiency electro-transformation of Escherichia coli with DNA from ligation mixtures Monique Jacobs, Stephan Wnendt and Ulf Stahl* Technische Universitaet Berlin, Fachgebiet fuer Mikrobiologie und Genetik, Seestrasse 13, D-1000 Berlin 65, FDR Submitted February 23, 1990
Transformation of E. coli by electroporation is a reliable method by which to obtain transformation efficiencies around 109 cfu/IAg, which are generally higher than those achieved by transformation via competent cell techniques (1). Here we present an improvement in the method which allows a more efficient transformation with ligation mixtures. A 200 bp cDNA fragment was ligated into the NcoI and PstI digested vector pKK233 -2 (2) under standard conditions and the ligation mixture used for electroporation of E. coli JM105 in a Bio-Rad Gene Pulser" (200 Ohm, 25 fiF, 10.5 kV/cm). Initial results indicated a very low transformation efficiency of the ligation mixtures. Since it has been reported that the presence of ions reduces the efficiency of electroporation (3) aliquots of the ligation mixture were dialysed for 45 minutes on microdialysis membranes (Millipore, type VS, pore size 0.025 Lm) against 10% glycerol/i mM EDTA, pH 8, prior to electroporation. As can be seen from table I, which shows the results of two independent experiments (assay A and control assay B), microdialysis of the ligation mixtures gives a 10 up to 700-fold increase in transformation efficiency. The reduced yield of transformants from the dialysed control in assay B compared to non dialysed control is due to the fact that the DNA can not be totally recovered from the filter. In contrast to microdialysis ethanol precipitation has no or little effect on the transformation efficiencies of ligation mixtures although the time constant is similar to dialysed probes (data not shown).
ACKNOWLEDGEMENTS We would like to thank Dr J.Woestemeyer and Dr A.Burmester for the kind permission to use their electroporation device and Dr M.Noyer-Weidner for providing strain K803. This work was supported in part by a DECHEMA grant to S.W.
REFERENCES 1. Dower,W.J., Miller,J.F. and Ragsdale,C.W. (1988) Nucl. Acids Res. 16, 6127-6145. 2. Amann,E. and Brosius,J. (1985) Gene 40, 183. 3. Shigekawa,K. and Dower,W.J. (1988) BioTechniques 6, 742-751.
*
To whom correspondence should be addressed
Table 1.Effect of ligation-buffer on transformation efficiency and time constant in electroporation. Control: 50 ng pAT153 Ligation mixture: 1 ng (1) or 5 ng (2) cDNA fragment ligated to 10 ng pKK233-2. In assay A strain JM105 was used, in assay B strain K803. DNA
number of transformants
time constant [ms]
assay A: control
1.8x 105
4.6
0 726 7 1450
4.1 4.6 4.2 4.6
3.6 x 07 2.9 x 105
4.6 3.3
1.9x 107
4.6
ligation mixture: untreated 1 dialysed 1 untreated 2 dialysed 2 assay B: control control in ligation-buffer control in ligation buffer and dialysed
Nucleic Acids Research, Vol. 18, No. 6
High-efficiency electro-transformation of Escherichia coli with DNA from ligation mixtures Monique Jacobs, Stephan Wnendt and Ulf Stahl* Technische Universitaet Berlin, Fachgebiet fuer Mikrobiologie und Genetik, Seestrasse 13, D-1000 Berlin 65, FDR Submitted February 23, 1990
Transformation of E. coli by electroporation is a reliable method by which to obtain transformation efficiencies around 109 cfu/IAg, which are generally higher than those achieved by transformation via competent cell techniques (1). Here we present an improvement in the method which allows a more efficient transformation with ligation mixtures. A 200 bp cDNA fragment was ligated into the NcoI and PstI digested vector pKK233 -2 (2) under standard conditions and the ligation mixture used for electroporation of E. coli JM105 in a Bio-Rad Gene Pulser" (200 Ohm, 25 fiF, 10.5 kV/cm). Initial results indicated a very low transformation efficiency of the ligation mixtures. Since it has been reported that the presence of ions reduces the efficiency of electroporation (3) aliquots of the ligation mixture were dialysed for 45 minutes on microdialysis membranes (Millipore, type VS, pore size 0.025 Lm) against 10% glycerol/i mM EDTA, pH 8, prior to electroporation. As can be seen from table I, which shows the results of two independent experiments (assay A and control assay B), microdialysis of the ligation mixtures gives a 10 up to 700-fold increase in transformation efficiency. The reduced yield of transformants from the dialysed control in assay B compared to non dialysed control is due to the fact that the DNA can not be totally recovered from the filter. In contrast to microdialysis ethanol precipitation has no or little effect on the transformation efficiencies of ligation mixtures although the time constant is similar to dialysed probes (data not shown).
ACKNOWLEDGEMENTS We would like to thank Dr J.Woestemeyer and Dr A.Burmester for the kind permission to use their electroporation device and Dr M.Noyer-Weidner for providing strain K803. This work was supported in part by a DECHEMA grant to S.W.
REFERENCES 1. Dower,W.J., Miller,J.F. and Ragsdale,C.W. (1988) Nucl. Acids Res. 16, 6127-6145. 2. Amann,E. and Brosius,J. (1985) Gene 40, 183. 3. Shigekawa,K. and Dower,W.J. (1988) BioTechniques 6, 742-751.
*
To whom correspondence should be addressed
Table 1.Effect of ligation-buffer on transformation efficiency and time constant in electroporation. Control: 50 ng pAT153 Ligation mixture: 1 ng (1) or 5 ng (2) cDNA fragment ligated to 10 ng pKK233-2. In assay A strain JM105 was used, in assay B strain K803. DNA
number of transformants
time constant [ms]
assay A: control
1.8x 105
4.6
0 726 7 1450
4.1 4.6 4.2 4.6
3.6 x 07 2.9 x 105
4.6 3.3
1.9x 107
4.6
ligation mixture: untreated 1 dialysed 1 untreated 2 dialysed 2 assay B: control control in ligation-buffer control in ligation buffer and dialysed
