Linear double-stranded DNA

Plasm id DNA (RFI) was linearised by incubation w ith site specific endonucleases in the buffers recommended by the enzyme supplier. The am ounts of enzymes required for complete cutting w ere determ ined in titration experiments. Standard incubation time was 90 m inutes, after which a sam ple was checked for complete digestion on a 0.8% agarose gel. Incubation tem perature was 37°C, except for Bsal which w as generally incubated at 55°C. Typical am ounts of restriction enzymes required to digest 1 pg of plasm id DNA in 90 m inutes were 1 U (Pstl), 2.5 U {BamHl, EcoRI, Hmdlll, Ncol) and 4.25 U (Bsal).

W hen linear DNA was prepared for 5' end-labelling, the restriction digest w as supplem ented after 90 minutes w ith 0.1 U calf intestine phosphatase per pg of DNA and incubation at 37°C was continued for another 30 m inutes. EDTA was then added to 50 mM and enzymes were inactivated w here applicable by heating to 65°C for 10 minutes. DNA was extracted w ith

Chapter 2

phenol/chloroform and chloroform (section 2.14), ethanol precipitated (section 2.15) and resuspended in TE. Linear duplex DNA w as stored at -20°C.

To generate substrates with four-nucleotide overhangs at the 5'end of the (-) strand for use in polarity experiments, pPB4.3 f(+) was linearised w ith either BamHl or Hindlll. Blunt ends were produced by treating these substrates w ith the Klenow fragm ent of DNA polymerase I in the presence of dNTPs. Duplexes w ith 3' overhanging ends were prepared by digestion of BamHI-linearised pPB4.3 w ith Sad and Hmdlll-linearised pPB4.3 w ith Kpnl. The 9 and 23 bp fragm ents generated in these digests were removed on Qiaquick spin columns, using the PCR prim er removal protocol supplied by Qiagen.

2.19 Single-stranded phagem id DNA

The bacterial strains JM109 (pDEA-7Z f(+)) and JM109 (pPB4.3 f(+)) were grow n on minim al m edia plates (M9) supplemented w ith 100 p g /m l carbenicillin. After 2 days at 37°C, single colonies were used to inoculate 10 x 2 ml cultures of Luria broth supplem ented with 0.1% (w /v ) glucose and 100 p g /m l carbenicillin. The cultures were incubated w ith good aeration until slightly turbid (approximately 2 hours). 150 |xl of helper phage (M13K07, 4.34 x 10^° p fu /m l) were added to each 2 ml culture and incubation was continued w ith good aeration for 1 hour. The phage-infected bacteria were then added to 4 x 2 1 Erlenmeyer flasks (4 ml of culture per flask) containing 400 ml prew arm ed Luria broth supplem ented w ith 0.1% (w /v) glucose, 100 p g /m l carbenicillin and 70 p g /m l kanamycin. The cultures were aerated vigorously for approx. 16 hours at 37°C. Cells were collected by centrifugation for 15 m inutes at 5000 rpm in a Sorvall GS-3 rotor.

The cell pellets were discarded and phage were precipitated by the addition of 40 g polyethylene glycol (8000) and 30 g NaCl per litre of supernatant. The suspension was stirred slowly for 1 hour at 20°C. The precipitate was collected by centrifugation for 20 m inutes at 8000 rpm in a

Chapter 2

Sorvall GS-3 rotor and resuspended in 25 ml TNE per litre of original supernatant. CsCl was added to a final concentration of 0.35 g /m l. The suspension was transferred to "Quick-Seal' polyallomer ultracentrifuge tubes, w hich were sealed and spun at 20°C, 55 000 rpm , for 24 hours in a Beckman Ti70 rotor. The phage particles appeared as a discrete band against a dark background and were removed from the centrifuge tubes using a needle and syringe.

Purified phage were dialysed against 2 x 2 1 of 50 mM disodium tetraborate (Na^B^O^) for 2 hours. The phage suspension was saturated w ith Na^B^Oy and transferred to a water bath at 70°C. An equal volum e of Na^B^O^- saturated phenol was also warm ed to 70°C and gently mixed w ith the phage suspension for 3 minutes. After a further 3 m inutes at 70°C the solution w as allowed to cool to 20°C and was centrifuged at 6000 rpm for 5 m inutes. The aqueous phase was collected and re-extracted twice w ith Na^B^O^-saturated phenol at 20°C. Traces of phenol were removed by dialysis overnight against 2 X 2 1 of TE at 4°C. The ssDNA was ethanol precipitated (section 2.15) and the concentration was determ ined as described in section 2.13. Single-stranded DNA w as stored in TE at 4°C. Typical yields from 1.6 1 of culture were betw een 0.6 and 1.5 mg of ssDNA.

2.20 Linear and fragm ented single-stranded DNA

0X174 ssDNA was fragm ented by a 20 seconds burst in a Heat Systems Ultrasonic Processor. Following this treatm ent the majority of ssDNA fragm ents were betw een 100 and 500 nucleotides in length as determ ined on a 1% agarose gel by comparison to molecular size standards (1 kb ladder).

For DNA binding experiments circular ssDNA (ssDEA-7Z f(+)) w as partially linearised by vortexing for 1 minute. Linear or fragm ented ssDNA w as 3' ^T-end-labelled using terminal transferase and [a-^^P]ddATP as described in section 2.24.

Chapter 2

2.21 N icked DNA substrates

To generate linear pDEA-7Z f(+) dsDNA containing a site-specific nick at the Pstl site, 180 pg of Psfl-linearised pDEA-7Z f(+) dsD NA were mixed w ith 250 pg pDEA-7Z f(+) circular ssDNA in 1 ml TE (pH 8.0) and dialysed for 12-16 hours against 2 1 TE (pH 8.0). The sample was divided into 4 equal aliquots and 200 pi H^O and 50 pi annealing buffer (lOx) were added to each tube. DNA was denatured and annealed by heating in a water bath to exactly 95°C for 5 m inutes followed by slow cooling to 20°C. Samples were then mixed w ith 125 pi agarose gel loading buffer (5X) and loaded onto 4 x 300 ml preparative agarose gels (1%) prepared in BRL Horizon 20x25 cm gel trays. Nicked circular dsDNA was separated from other DNA forms by electrophoresis at 200 V for 3 hours at 4°C. A 2 cm slice was removed from either side of the gels and stained in TAE containing 2 p g /m l ethidium bromide. The position of the nicked DNA w as identified using a UV transilluminator and served as a reference to excise the complete band in the absence of UV.

The gel slices containing nicked circular DNA were placed into dialysis tubing containing approximately 5 ml TAE buffer. Air bubbles were rem oved and the dialysis tubing was sealed. DNA was recovered from the gel slices by electro-elution at 50 V in BRL Horizon 20.25 gel tanks containing TAE buffer at 4°C. After 12 hours the current was reversed for 3 minutes. Buffer containing nicked circular DNA was collected from the dialysis bags and concentrated by ethanol precipitation (section 2.15). DNA pellets w ere dissolved in TE and dialysed against 2 1 TE for 12-16 hours. DNA concentrations were determ ined as described in section 2.13. Generally around 40 to 50 pg of nicked circular DNA were recovered.

Circular pDEA-7Z f(+) DNA (10 pg), containing a site-specific nick at the Pstl site, was linearised by incubation with Bsal (50 U) at 43°C. After 90 m inutes an additional 30 U Bsal and 10 U calf intestine phosphatase were added, followed by incubation for a further 30 minutes. Bsal w as not used at its optim al

Chapter 2

tem perature (55°C) to avoid dissociation of the 174 nucleotide fragm ent betw een the Pstl and Bsal restriction sites. Nicked linear duplex DNA was purified on Qiaquick spin columns and 5' ^^P-end-labelled as described in section 2.23.

Nicked linear pPB4.3 DNA was prepared essentially as described for pDEA-7Z. Form I DNA was digested with either Ncol or BamHl, denatured and annealed w ith pPB4.3 ssDNA to form nicked circular DNA. Circular DNA, containing the nick at the BamHl site was linearised by digestion w ith Ncol, dephosphorylated and then 5' ^^P-end-labelled (section 2.23). The com plem en­ tary substrate containing a nick at the Ncol site was digested w ith BamHl and 3' ^^P-end-labelled as described in section 2.24.