Reply to Fast& Cheap Plasmid Miniprep Kit
bernard at elsie.nci.nih.gov
Fri May 30 15:35:15 EST 1997
In article <5mm5qs$ml at bioalp.biobase.dk>, wind at biobase.dk says...
>Bernard Murray (bernard at elsie.nci.nih.gov) wrote:
>: For sequencing I double-load (3 ml culture) and clean the
>: crude plasmid with PEG/NaCl which seems to work as well as phenol
>: but is cheaper and less nasty. This is for manual sequencing with
>: Sequenase so I don't know how automatics would cope. This fails so
>: seldom (does *anything* work 100% of the time!?) that I'll not
>: bother looking at anything else.
>Bernard, could you give some details on the PEG/NaCl alternative to phenol?
This is just an abbreviated version of the protocol described in
Sambrook et al. (page 1.40 in my copy). I dissolve the final pellet
from the crude prep in TE/RNase and incubate a short while then;
Take the fairly crude plasmid (in TE) eg. 20 ul
add half a volume of 5 M NaCl eg. 10 ul
add 1.5x the plasmid volume of 13% PEG 8000 eg. 30 ul
leave on ice for 20 minutes (my habit)
spin in a microfuge at full speed for 5'
dump the supernatant as cleanly as possible
wash once with 70% ethanol (eg. 1 ml)
resuspend in TE (eg. 20 ul)
sequence (eg. an equivalent of 0.75 - 1 ml of starting culture)
This is adequate for my purposes, which is for relatively short reads
(as I mentioned before I'm using plain Sequenase). I'm not saying this
will be as good as phenol extracted plasmid for all downstream techniques
(ie. don't blame me...). If I want to do long reads I go the whole hog
and band it with CsCl but I like to use the above techniques to narrow
the selection of clones to play with.
I hope that this is of some help,
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
(but soon moving to San Francisco)
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