'Cracking'

Bruno Cenni cenni at cellbio.unige.ch
Wed Jun 9 02:09:37 EST 1999


Hi Robert,

this protocol looks differences in the size of uncut plasmids (you'll be able
to see inserts as small as about 2-300bp). I didn't find the ref. anymore...

After transforming the bugs with your ligation and growing them on a plate
o/n:

-Number colonies on the plate
-With a sterile pipet tip pick colonies and resuspend them each in a tube
with 12 ul Bug-Mix
-Mix, let sit at RT for 5-10 min.
-Add 8 ul of phenol/chloroform (Tris-buffered phenol)
-Vortex a few secs.
-Spin in eppi fuge at max. speed 5-10 min.
-Load upper aqueous phase onto thin agarose gel
-Look at supercoiled ligated plasmids vs empty vector

Bug-Mix:

0.5 mg/ml lysozyme
25 mM EDTA pH 8
25 mM Tris HCl pH 7.5
0.1 mg/ml RNase
10% v/v glycerol
few crystals bromophenol blue

aliquots at -20

Good luck

"Dr. Robert Gay" wrote:

> Dear all
>
> We're looking for a protocol for the rapid screening of plasmid DNA
> directly from transformed E.coli colonies (avoiding the overnight liquid
> culture step).
>
> It's a protocol you may know as 'Cracking'.  We had such a protocol, once
> upon a time, but have  lost it !
>
> Hope someone can help.
>
> Thanks in advance
>
> Robert
>
> Dr Robert Gay
>
> Post Doctoral Research Fellow
> CRC for Biopharmaceutical Research
> Department of Biotechnology
> University of New South Wales
> Sydney, NSW 2052, Australia
>
> Tel : +61(0)2-9385-1892
> Fax : +61(0)2-9313-6710




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