DE52 Lambda Phage DNA prep

• 1st, prepare a stock of DE52 DEAE cellulose (Whatman)
• Add ~15 gm resin to 500 ml 0.5N HCl - mix, leave to settle - 30'
• Pour off acid and any resin "fines"
• add 500 ml TEM buffer, PH 7 (TE + 20mM MgCl₂ - important!)
• Mix, allow to settle - or spin down (5K/5')
• Repeat TE washes until pH of Resin is 7
  This can take 6-7 spin/washes - you can speed this up by using 10xTE for the first couple of washes (better buffering out of the acid)
• Spin down resin - resuspend at 75% resin + 25% (volume for volume) TEM
• Store at 4° (add NaAzide if you need to keep it a long time)
• Mix well before each use

For a small scale prep (scale up if needed)

• Prepare a good high titre plate, or liquid phage lysate (see "Maniatis Manual")
• Mix 600µl of phage lysate + 600µl of the DE52 slurry in a microfuge tube
• Mix well, spin ~3K/2'
• Transfer supernatant to fresh tube and repeat the DE52 step 3 more times (The DE52 is binding out the E. coli nucleic acids, proteins, oligosaccharides, etc)
• Spin supernatant once more (10K/1') to pellet & remove any residual DE52 (important - otherwise this would bind out the phage DNA when the particles are burst)
• Transfer 800µl to a fresh tube
• Add 100µl 5M NaCl + 540µl isopropanol, mix, put at -20° for 1 hour (coffee time)
• Spin down particles at ~12,000 rpm/10' in microfuge
• Resuspend particles in 500 µl 70% ethanol (nb they are not easy to see at this point - likely to be smeared down the tube wall so be careful)
• Re-spin
• Re-suspend particles in 200 µl TE (no MgCl₂)
• Phenol extract (to burst the phage), CHCl₃ and ethanol ppt. the phage DNA in the usual way

For gt10/gt11, assuming you have good starting lysate, this gives enough DNA for 3 or 4 digests which provides ample material for subcloning the insert.

Using this method I have never had DNA that doesn't cut the 1st time. If your yields are low, your starting lysate was too low in titre.