simple protocol for colony PCR

[Robert...@jcu.edu.au]

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 need such a protocol for work in a developing nation.

Thanks in advance

Cheers,

Robert

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Dr Robert J. Coelen fax: 61-77-791 526
Dept of Biomedical and Tropical Veterinary Sciences phone: 61-77-815 024
James Cook University of North Queensland
Townsville-just-about-on-THE-reef, Q, 4811
-----------------------from the land d r---------------------------
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[Anthony Palombella]

Robert...@jcu.edu.au writes:

>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 need such a protocol for work in a developing nation.

I have successfully PCR'd from both coli genomic DNA and plasmid DNA by
picking a colony from a plate with a sterile toothpick or P-20 tip and
resuspending the colony in a standard PCR reaction mix. In my hands, it
often takes two rounds of amplification: I see little product after 30
cycles or so, but using ca. 5 ul of that reaction in a new reaction gives
me good results. In the first reaction expect to see chromosomal DNA and
RNA on the gel. It takes very few cells for this to work, much less than
an entire colony. Good luck!

-- Tony
--
>>>>>>>>>>>>>>>>>>><<<<<<<<<<<<<<<<<<<<
>>Ehh... I could be wrong, ya know...<<
>>pal...@beagle.colorado.edu <<
>>>>>>>>>>>>>>>>>>><<<<<<<<<<<<<<<<<<<<

[Sebastian W. Bunka]

Robert...@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
--

email: [ Sebasti...@vu-wien.ac.at ]
voice: FAX:
+43-1-71155260 +43-1-7149110
Location: earth, europe, austria, vienna Inst. of Bacteriology Vet.Univ.

[Ferenc Marincs]

In article <385bkr$o...@osiris.wu-wien.ac.at>,
Sebasti...@vu-wien.ac.at wrote:

I usually resuspend some colonies from the agar plate in 50 ul of water
and use 5 ul form that suspension in the 100 ul PCR reaction without any
treatment. 5 ul bacteria from an overnight cultured broth works also very
well in my hands.
Good luck, Frank

Ferenc Marincs
AgResearch, Private Bag 11008, Palmerston North, New Zealand
Phone: +64-6-356 8019, Fax: +64-6-351 8032
E-mail: agp...@pnv.palm.cri.nz