Gene. 46 (1986) 287-290 Elsevier GENE 1731 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Short Communications RNA colony hybridization method (Recombinant DNA; in situ hybridization; gene transcription; oligodeoxynucleotide probe) Ivan Ivanov and Lilly Gigova institute of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Molecular Biology, Bulgarian Academy of Sciences, II 13 Sofia (Bulgaria) Tel. 72-41-53 (Received March 12th, 1986) (Revision received May 7th, 1986) (Accepted May 14th. 1986) 287 SUMMARY A method for rapid screening of specific RNA sequences in recombinant colonies by hybridization in situ is presented. The method includes two consecutive steps of lytic treatment of the nitrocellulose-filter-supported colonies (10% sodium dodecyl sulfate and 3 x SSC at 65 “C) and hybridization with 32P-labelled specific oligodeoxynucleotides. INTRODUCTION The method of DNA colony hybridization has been introduced by Grunstein and Hogness (see Grunstein and Wallis, 1979) and it turned out to be a useful method for rapid screening of colonies containing specific DNA sequences. In this paper we present a similar (in respect to technical perform- ance) method allowing rapid screening of colonies for specific RNA sequences by in situ hybridization. Abbreviations: bp, base pair(s); L-broth, Luria broth; nt, nucleo- tide(s); oligo, oligodeoxynucleotide; SDS, sodium dodecyl sul- fate; SSC, 0.15 M NaCI, 0.015 M zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Na, . citrate, pH 7.6; Tc, tetra- cycline. EXPERIMENTAL AND DISCUSSION (a) Growth of bacteria and treatment of the colonies (fmal protocol) Recombinant bacteria Escherichiu coli K- 12 (LE392) containing a synthetic human calcitonin gene inserted in expression plasmids (see section c) were transferred with sterile toothpicks to nitrocellu- lose filters placed on L-agar petri plates (with 10 pg Tc/ml) and grown overnight at 37 ‘C. The filters with the colonies were transferred consecutively to two petri dishes containing filter paper discs presoaked with 10% SDS (3 min at room temperature) and 3 x SSC (15 min at 65°C). The filters were air-dried at 37’ C for 10 min, bak- ed at 70 oC in a vacuum oven for 15 min and used for hybridization. 0378-l 119/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)