Dan Kalderon

SMALL-SCALE PREPARATION OF BACTERIOPHAGE LAMBDA DNA

small-scale DNA prep; bacteriophage lambda DNA; lambda DNA;
Growing Up Phage
DNA prep.;
Plate lysate
 Make lambda agarose plates; (preferably in NZCYM, although LB+ 10mM MgSO4 is OK; 1.5% agarose).Add 20µl (gt10) or 200 µl (Charon 4) of single plaque eluate (from 2ml in TM10+chloroform) to 100µl of indicator bacteria in TM10, mix, incubate 20' at 37°C and spread on a dry 9cm agarose plate in soft agarose (0.7% 48°C).Incubate for about 12 hr. Optimally the plaques should just touch each other after about 8 hr but the yield of phage is reduced less by adding too much than too little phage. As lysis is normally complete it is best also to have one plate with no added phage to be sure that the plates could support a good lawn of bacteria. Add 5 ml of TM10 to each plate and elute at room temperature for at least 2 hr (or O/N at 4°C) with gentle agitation. Collect eluate, adding a further 2 ml TM10 to maximise yield. At this point a significant proportion of phage still remains on the plate and can be eluted with a further, say, 5ml of TM10+gelatin to give high titre phage stocks(stored with a little,0.2%,chloroform at 4°C)

Liquid culture
 Plate lysates are preferred to liquid culture because fairly reproducible yields of phage are usually obtained without accurate determination of phage titre. Nevertheless , some people find it convenient to grow 50-100ml liquid cultures by innoculating with 1ml of a C600 O/N per 100ml NZCYM and infecting with 0.5-2 X 107 phage (more with Charon 4 than gt10). Grow for 4-7 hr or until lysis occurs.

Preparation of DNA

1) Spin down bacterial (and other) debris from plate lysates or liquid culture (5000 rpm 5' Sorvall, 10 ml polypropylene tubes).

2) Add RNase A & DNase I each to 1µg/ml to supernatant and incubate at 37°C for 30'.

3) Add an equal volume of 20% PEG (polyethylene glycol),2M NaCl in TM10, mix and stand on ice for at least 1-2 hr.

4) Spin down phage (7000 rpm 20' Sorvall SS34), pour off liquid, spin again for 30 secs and aspirate fluid.

5) Resuspend pellet thoroughly in 0.5ml TM10 and transfer to a microfuge tube.

6) Extract with 0.5ml chloroform

7) Add 5µl 10% SDS, 10µl 250mM EDTA pH 8 and incubate at 65-70°C for 10mins.

8) Add 0.5ml phenol (equilibrated with TE) and shake to mix (but do not vortex henceforth); centrifuge for 2 min and remove aqueous phase which will probably drag some interphase along due to high viscosity.

9) Extract with phenol/chloroform/iso-amyl alcohol (25:25:1).

10) Add 0.5ml isopropanol and mix. A DNA precipitate should form but if not, leave at -70°C for >15 min, thaw and centrifuge. If DNA precipitate is readily visible spin for only a minute (to help subsequent dissolution), otherwise 10 min. Wash pellet in 70% ethanol, dry and dissolve in 50-200µl TE by gentle shaking or with the aid of a Gilson. The yield of gt10 recombinants is usually greater than for Charon 4 but the latter normally gives enough DNA for at least 5 restriction enzyme digests. As a large amount of RNA is normally present in the DNA preparation it is worth treating with boiled RNase A (at ~100µg/ml) during or prior to digestion, so that (i) small DNA fragments are not obscured and (ii) the size of fragments can be more accurately estimated from their electrophoretic mobilities. (In the presence of excess RNA, all fragments will migrate faster than they should).