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We describe here a procedure for in situ bacterial colony lysis that gives much more intense hybridisation signals which do not change significantly upon reusal of filters. This is achieved by proteolysing the debris with proteinase K (without shaking) followed by blotting the DNA down onto the nylon membrane. The procedure is a much simplified version of the original experiment described in (1) with the addition of the steaming step (2) in alkali which contributes to the signal increase. The specific signal in Fig IB is 10 to 50 fold higher than in the conventional colony lysis (Fig 1A), and the relative signals of positive and negative clones don't change upon rehybridisation (1C). The increase in the signal (IB compared to 1A) brings the mass of DNA extracted from the colony over the critical threshold for detection with oligonucleotide probes as short as 11 bases (Fig. 1). Specific hybridisation of short oligonucleotides to clones has a number of applications (4—6). The shortest oligonucleotides thus far hybridised in situ to cosmids were 12 bases long (3). By enabling hybridisations of 11-mers (even 10-mers, DN & RD unpublished) to cosmids, the procedure described in this report plays an essential part in the oligonucleotide fingerprinting approach for the construction of ordered clone libraries and facilitates image analysis of high density cosmid or plasmid lifts by avoiding smeared signals.

An improved bacterial colony lysis procedure enables direct DNA hybridisation using short (10, 11 bases) oligonucleotides to cosmids