phage prep help really, really needed
clemens at eichow.tuwien.ac.at
Fri Nov 11 10:28:05 EST 1994
deleted to save space
I used both liquid lysate (prepared acc. to Promega Applications Guide,
basically the same as Sambrook at al.) and plate lysate, with the difference
to your procedure that I scraped the top agarose off the plates, breaking it
to little pieces with a spatula, then shaking it with 2,5 ml of Phage buffer
per plate (90mm plates) for approx. 1 hr, then spinning the agarose clumps
down. Gives fairly good yield.
Precipitation of the phage: Dunno what could be wrong, I did it basically
the same way as you did.
After that, CHCl3 extraction (rids you of residual PEG). Aquaeous phase
treat with RNase & DNase, 30` 37°C. Add EDTA and SDS (to 0,02M
and 0,1% resp.), 15 `68°C. EXtract w/Phenol-CHCl3 and CHCl3.
Add 0,1x Vol 5M NaClO4 & 1x Vol Isopropanole, 45` -20°C, spin down.
Dissolve in 400 ul TE, ppt w/0,1x Vol 3M NaOAc & 2,5x Vol EtOH,
mix carefullly, the DNA will form a viscous cloud you can "grab" with a
glass hook. Put it in 70% EtOH to wash, spin down, dry. If you start with
enough lysate (enough phage titre, i. e.) this shpuld give you enough
very clean DNA.
Alternative: the Promega Magic (they are called Wizard now, I believe)
lambda prep kits, or the QIAGEN lampda prep kits. Both work fine, you
just have to prepare a good lysate.
Good luck, Clemens
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