single colony plasmid isolation

[Juergen Soutschek]

Dear Netters,

I want to do single colony sequencing and therefore selected two
protocols for isolation of plasmid DNA from a single bacterial colony.

protocol #1
- suspend colony in 25ul TE
- + 1,25ul of 1mg/ml ProtK; 15' 55 deg. C
- 15' 80 deg.C, 1' ice, pellet debris 2' at 10,000 rpm

protocol #2
- suspend colony in 25ul TE
- 5' 100 deg.C, 1' ice, pellet debris 2' at 10,000 rpm

Is the gained amount and the purity of the DNA sufficient for cycle
sequencing?
Besides I'm not sure about the Proteinase K step. Is it necessary?
Any suggestions or experiences ?


-- 
Juergen Soutschek
sout at gen.mpib-tuebingen.mpg.de

Max-Planck-Institute for Developmental Biology
Department of Physical Biology
Spemannstr. 35/1

72076 Tuebingen
Germany