l phage DNA preparation

BULK GROWTH OF l PHAGE AND PURIFICATION OF ITS DNA

Day 1

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 + P2392)
  
  
• 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.

Phage purification
 

• 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