DE52 Lambda Phage DNA prep

john brennand john.brennand at gbapr.zeneca.com
Fri Feb 16 07:30:33 EST 1996


Several people have asked me for details of this protocol and I am having 
problems with my E-Mail - so, here it is:-


        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 4o (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 600ul of phage lysate + 600ul 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 800ul to a fresh tube
Add 100ul 5M NaCl + 540ul isopropanol, mix, put at -20o for 1 hour (coffee 
time)
Spin down particles at ~12,000 rpm/10' in microfuge
Resuspend particles in 500 ul 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 ul 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 1st time. If your 
yields are low, your starting lysate was too low titre.

John





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