A fast and convenient way to produce single stranded DNA from a phagemid

Despite the increased number of methods using double stranded DNA for sequencing, site directed mutagenesis, e t c . , it is often more convenient to use vectors able to produce single stranded DNA for such usages. The phagemids are more convenient for many purposes in molecular biology than M13 phage because they are smaller and can grow on bacteria as colonies instead of plaques, However the obtention of single stranded DNA from phagemids is longer than from M13 since it needs an infection step with a helper phage. A convenient protocol for single stranded DNA production from phagemids is proposed here. Used with pEMBL8 and derived vectors, this method provides similar yields than other described methods (1-3). It avoids absorbance monitored culture, saves up to one day and is far less consuming in helper phage stock. A colony of male E. coli strain harboring the phagemid of interest, taken from a fresh selective medium plate (less than one week-old), is suspended in 10-50 /xl of 2YT medium. The bacterial suspension is then infected by addition of 1 y\ of M13 KO7 helper phage stock (more than 10 pfu/ml) and incubated 15 min at RT. 500 /tl of 2YT with antibiotic for phagemid selection are added to the infected cells which are then incubated at 37 °C for one hour to let helper phage express antibiotic resistance. 100 to 200 /tl of incubated cells are added to 3—4 ml of 2YT medium with appropriate antibiotics (we used 150 mg/1 ampicillin and 75 mg/1 kanamycin), The culture is proceeded for 18-20 hours at 37°C. The culture should be lead to saturation for good single strand production. The virion particles are isolated and single stranded DNA is prepared as in (3). 1.5 ml culture leading to 30 /tl of preparation should provide concentrations suitable for most applications (see figure).