Particle bombardment

Gerd Nilsen gerd.nilsen at biotec.no
Thu Nov 17 03:11:58 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
....
> > > Sharp Tool
> >
> > Hi,
.....
> > 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
> ------------------------------


I used to bombard with plasmid isolated by commersial kits. Depending on the plasmid, the kit and the carma of the day, I would get 30-200 ng/μl. (Diluted to taste after quantification)
I do not like Qiagen miniprepkits, because of low yield and high cost, but was very satisfied with Bio-Rad kit. 

I have never in my long life obtained 1 μg/μl or more from any miniprep (precipitation and redissolvin of cause give you any conc you want), 
- and have concluded that people who get that must have special skills, either in isolating, or in meassuring DNA content. Some of the same people also load 2-3 μl of these 1 μg/μl preps on a normal agarosegel, and get it all separated. 

According to Maniatis and most other lab-guides, 200 ng is roughly the limit of separation for a normal well in a normal gel. That of cause depends on the size of comb, and my experiments show that 0.75 mm x 5 mm comb must have been used when this estimate was done.

For quantification, I really was satisfied the day I could buy DNA ladders with quantified bands, which could be used to quantify DNA samples. I bought them from Fermentas, but I guess other companies have also put such in their catologues by now. 

mvh Gerd




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