BULK GROWTH OF l PHAGE AND PURIFICATION
OF ITS DNA
Phage plate stock
Incubate for 20 mins at 37°C 50µl of a screening positive pure
plaque pickate (plaque/agar plug in 0.5ml of SM and 20µl of chloroform),
50µl overnight E. coli (appropriate strain). (Half these amounts
for a miniplate).
Plate on 50mm LB agar with 0.9ml LB top agarose and incubate inverted for
4-6 hours at 37°C by which time the bacterial lawn should have completely
lysed. If the lawn does not grow then replate with less pickate and if
the lawn does not completely lyse then replate with more pickate.
With a bent yellow tip, scrape off the top agarose into a 1.5 ml microtube
and top up with SM and add 50µl of chloroform. Vortex mix and
leave in the fridge for at least 2 hours.
Spin the tube for 5 mins at full speed and pippette of the supernatant
to sterile tubes. This is the plate stock and should have a titre of around
109pfu/ml. Can store indefinately at 4°C.
Alternatively collect 6-10 positive plaques and pool in 0.5ml of
SM and 2oµl chloroform and use this as a stock.
Bulk phage growth
Incubate 1-100µl (depends on phage, titre and host strain- can use
1, 10 and 100µl of same stock simaltaneously and only prep fully
lysed cultures) of l plate stock to 200µl
of overnight E. coli appropriate host strain, for 20 mins at 37°C
in a 50ml Universal. (lgt11 + C600 or Y1090;
lZAP + XL1Blue; lFIX/lEMBL
Add to this 20ml LB medium containing 5mM MgCl2 and continue incubating
at this temperature for 4-8 hours with vigorous shaking. For larger preps
the amount of medium can be increased and the incubation shaken in sterile
flasks. The culture should lyse during this time and be seen to clear and
be full of ‘bits’. If this does not happen, start again with more phage.
The exception is lgt11 in E. coli C600 which
does not appear to lyse but still produces plenty of phage in the medium.
Add 20µl of 20mg/ml RNAse A and 20µl of 80000U/ml crude DNAse
and incubate for 20min at 37°C.
Add 0.5ml of chloroform and vortex vigorously.
Complete sterility is no longer necessary. Transfer the culture to a Oak
Ridge polypropylene centrifuge tubes and spin at 10000rpm (10000g) in the
benchtop Jouan for 5mins.
Transfer supernatant to another centrifuge tube and spin at 21000rpm in
the Sorvall for 1 hour at 4°C.
Resuspend the small translucent phage pellet in 400µl SM by whirlimixing
vigorously and transfer to a microtube.
Extract with an equal volume of chloroform. Recover supernatant. If there
is a good phage yield the supernatant should be slightly milky.
Add 10µl of 0.5M EDTA and 20µl of 5M NaCl for every ml of phage
suspension and mix well. Extract once with an equal volume of phenol by
mixing carefully. Spin and collect supernatant and extract with an equal
volume of chloroform. Spin and collect supernatant, add 2 vols of ethanol
and precipitate on ice for 15mins. A fluffy DNA precipitate should be visible
if the prep is good.
Spin at full speed in a microcentrifuge for 10mins and wash the pellet
by adding 1ml of ice cold 70% ethanol for 2min. Tip this out, spin briefly
to collect the excess ethanol, pippette off carefully and allow to dry
on the bench for a few mins before resuspending l
DNA in 20-100µl of water.
LB media; 10g tryptone, 5g yeast extract, 5g NaCl
LB agar; LB media with 10g/l agar
LB top agarose; LB media with 0.6g/100ml agarose
SM 10mM TRIS/HCl, pH 7.4, 10mM MgSO4, 0.01% gelatin