100, 243-255. 4. Sambrook,J., Fritsch.E.F. and Maniatis.T. (1989) Molecular
Cloning: A. Laboratory Manual. (2nd edition). Cold Spring Harbor Laboratory
Press,.
© 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 23 6655
A rapid procedure for the screening of recombinant plasmids C.Le Gouill and C.V.Dery* Departement de Biologie, Universite de Sherbrooke, Sherbrooke, Quebec J1K 2R1, Canada Submitted June 24, 1991 The screening for recombinant plasmids can be a time-consuming task when no selection or colorimetric detection of recombinant over intact plasmids can be used. Some techniques were developed to analyze recombinant plasmids (1, 2), but they yield problems of desiccation of the samples, high viscosity of the DNA preparation or contamination by chromosomal DNA. In this work, using some solutions of the alkaline lysis procedure (1, 3), a rapid one-tube technique was developed for the analysis of recombinant plasmids with which the samples are applied directly on the gel. Transformed bacteria were plated on Terrific Broth (4) plus 1.5% agar and the desired antibiotic and were generally incubated overnight. To 1.5 ml microcentrifuge tubes, 16 /il of lysis solution was added. The lysis solution contained 9 vol of 10 Xgel loading buffer (10X: 0.25% bromophenol blue; 0.25% xylene cyanol, 25% Ficoll type 400) (1), 11 vol of water and 40 vol of solution 2 (0.2 N NaOH, 1% SDS). Then, with an automatic pipette, individual colonies (1.5—2 mm diameter) were harvested by pressing the tips (20—200 /tl) in the colonies and were inoculated (just touch the solid medium) on a plate in an ordered array for bacterial storage and recovery. The rest of the bacterial colonies inside the tips were resuspended in the lysis solution by pipetting gently 4 - 5 times. It is important to avoid the formation of bubbles. Then 3 /il of solution 3 (3 M potassium acetate, 1.8 M formic acid) (3) was deposited on the wall of the microcentrifuge tubes. The samples were centrifuged for 4 min and the supernatants (up to 16 /*1) were applied onto individual wells of a submerged horizontal agarose Tris—borate—EDTA gel (1). After electrophoresis, the gel was stained with ethidium bromide and photographed. When the diameter of the colonies was smaller than 1.5 mm, the colonies were harvested by suction and the volumes of the solutions were reduced to half. All the solutions were stored at room temperature and all the manipulations were carried out at room temperature. In order to determine the efficiency of this technique, we compared it with published procedures (1, 2) using plasmids of different sizes (Figure 1). For unknown reasons, the 4.7 kbp plasmid always migrated as a doublet with the procedure described in (2). With our method, only negligible amounts of chromosomal DNA were detectable and plasmids as large as 14.7 kbp were easily detected. Even though no RNAse treatment was used, RNA was also negligible and did not interfere with analysis of small plasmids. As our method does not require any incubation, the routine preparation of plasmid DNA of 24
colonies can be performed in 10 min. The solutions used are the same as the alkaline lysis method (1,3) and there are no problems related to the viscosity of the DNA preparation for the loading of the samples on the agarose gel. Solid Terrific Broth medium (4) was used instead of LB agar (1) because the bacteria grow more rapidly and the colonies can be harvested as soon as 12 h after plating. REFERENCES 1. Maniatis,T., Fritsch,E.F. and Sambrook.J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 2 Shepard.B.A. and Shaffer.J.B. (1990) BioTechmques 8, 388. 3. Birnboim.H.C. (1983) Methods Enzymol. 100, 243-255. 4. Sambrook,J., Fritsch.E.F. and Maniatis.T. (1989) Molecular Cloning: A Laboratory Manual. (2nd edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
B 1 2 3 4
4 3 2 1
1 2 3 4
Figure 1. 0.8% agarose gel electrophoresis of plasmid DNA isolated as described in ref. 2 (A), in ref. 1 (B) and in this work (C). Lane 1: 14.4 kbp; lane 2: 4.7 kbp; lane 3: 3.2 kbp; lane 4: 2.7 kbp.
* To whom correspondence should be addressed
Downloaded from https://academic.oup.com/nar/article-abstract/19/23/6655/2387404/A-rapid-procedure-for-the-screening-of-recombinant by guest on 20 September 2017
