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Cracking Preps for identifying inserts

Greg Nattrass gnattras at waite.adelaide.edu.au
Sat Aug 29 06:39:26 EST 1998


COLONY CRACKING

A great method for analysing recombinant E. coli clones. This technique
is less labour intensive and faster than minipreps and colony PCR. 

The lysis buffer contains the following.   
                    NaOH (2M)           	    25ul
                    SDS (10%)              	    50ul
                    EDTA (0.1M)          	    50ul
                    10x glycerol load buffer    50ul
                    Water               		825ul

I run 10ml 1% agarose gels on double width microscope slides. The thin
agarose layer concentrates plasmid DNA, a DH5a colony generally has 50ng
of supercoiled vector. 

15ul of the lysis solution is added to a number of eppies. An individual
colony is picked from LBamp plate with a yellow tip. Gently streak the
tip on a master plate and place it in the lysis solution. Repeat this
process for each colony to be analysed. Leave the tip in the lysis
solution and give each tube a vortex then incubate at 65C for 15 mins.

Load ~5ul into each well and run samples into the gel under low voltage
(~30V). At this point add more TAE buffer  so the wells are now
submerged and increase the voltage to 80V. Run the bromophenol blue to
the bottom of the gel. I run MW markers in lane 1 and a colony from
vector religation in lane 2. The remaining 11 lanes contain possible
recombinant clones. Remember the plasmids are supercoiled so you  must
run the vector only colony to accurately size insert containing
colonies.

This procedure works excellent with small colonies, there is no need to
use bacterial streaks as too much protein will inhibit the agarose
separation. I have identified inserts in pBluescript as small as 200bp.
The pBluescript vector only colony usually runs with the first doublet
(1.95/1.86kb) SPPI/EcoRI markers. Once insert containing clones have
been identified, pick the corresponding colony from the master plate and
setup a culture(s).

I think this is the reference for the technique. 
Plasmid detection and sizing in single colony lysates
Barnes-WM (1977). Science 195(4276). 393-394. 

Any further questions feel free to email me again.

Greg Nattrass



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