3 replies to this topic

[Hiprum]

Hi there,

I'm looking for a protocol to directly extract DNA from E.Coli (DH5Alpha) colonies on Agar+ Amp plates without growing them overnight in LB broth. The reason for this is that while the bacteria grows well on the plates it does not do so in the broth, so we're trying to side step this problem. Any suggestions?

Thank you

HipRum

[HomeBrew]

If the plates are struck from a single colony and are thus all the same, you can make a "fake overnight" by suspending an amount of the lawn in L-broth or a buffer.  Centrifuge to pellet, and proceed as if the pellet was from a true overnight culture.

[pDNA]

you can also try to use different media ...if your gene is toxic and under the control of a e.g. IPTG inducible promoter try to grow the cells with addition of glucose in LB or use M9 minimal medium. Maybe this helps. If u use a T7-based expression systems there are plasmids available (pLys) that reduce basal transcription from the T7 promoter.

Regards,
p

Edited by pDNA, 14 January 2011 - 11:53 PM.

[Michaelro]

Hi
If you still certain that you want to extract DNA from the colonies - we've used Templifee kit from Amersham couple of years ago.
It allows you to prepare sufficient amount of DNA for sequencing.
Hope it works for you.
Good Luck
Michael