simple protocol for colony PCR

[Sebastian W. Bunka]

Robert.Coelen at jcu.edu.au wrote:
: Hi,

: Could anyone give me a simple protocol for direct PCR of plasmid DNA from 
: bacterial colonies, taken directly of the plate. The emphasis is on simple !
I didn't try it with plasmid dna, but for amplification of a genomic
fragment:
Grow overnight on (in my case blood agar), 
strain Actinobacillus spec.; pick some colony material and dissolve
in distilled water (OD660 0.1-0.25 about 10E8-9 bugs, boil for 10 minutes;
use 2 microliter in 50 microl. of your PCR setup; for my gene I run
the PCR with 25 cycles (40 sec. 94 dC, 40 sec. 50 dC, 90 sec. 72 dC, and
a last cycle 5 min. 72 dC) when I run 10 microl. on a gel I have nice
visible bands at 1700 bp).

Hope this helps, Sebastian
--

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