Particle bombardment

Sharp Tool sharp.tool at bigpond.net.au
Tue Nov 15 05:49:16 EST 2005


> Sharp Tool schrieb:
>
> > Hi,
> >
> > Normally a 0.5 - 1.0 ng/ul of purified plasmid DNA is used for particle
bombardment.
> >
> > QUESTION: Does anyone use a commercial miniprep kit to purify plasmid
DNA from an overnight culture to achieve this concentration? I found that
the yield is very low, and no way of getting this concentration unless I
precipitate the DNA and resuspend it in a smaller volume.
> >
> > Sharp Tool
>
> Hi,
>
> I'm not sure whether I understood your problem right, but there
> shouldn't be any problem to reach the concentration you mentioned.
> What are the bacteria and plasmid you use? Do you have to use special
> bugs or plasmids for your experiment, or can you use the "usual" stuff?
>
> When I use the common alcaline miniprep isolation protocol on 4 ml O/N
> culture (XL1-Blue, multi-copy plasmid), I usually end up with 100 ul at
> 1-1.8 ug/ul.
>
> Cheers,
> Klaus

I take it your not using a commercial kit (e.g., a QIAGEN miniprep kit) -
They give low yield.
Your concentrate is about 40X higher than mine - impressive.

By the way, how to tell if a plasmid is a low copy vs high copy by looking
at the plasmid map?

Cheers
Sharp Tool





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