eimert at web.de
Fri Nov 18 05:36:26 EST 2005
Gerd Nilsen schrieb:
> 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/ìl. (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 ìg/ìl 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 ìl of these 1 ìg/ìl 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
I can assure you, that 50 ug plasmid (pGEM-T Easy in XL1-Blue, for
instance) from 5 ml O/N culture is nothing special with the Maniatis
(slightly modified) protocol.
I know, that concentration determination by UV tends to give an
overestimation, so I use the same Fermentas ladder to verify - there is
no huge deviation.
Also, in my experience the 200 ng resolution limit for a 5 mm well
doesn't hold true in our lab practice (btw, Maniatis states 500 ng per
5 mm well). Did you actually try that by yourself? For instance, when I
use 20 µl of the Fermentas ladder in a 4 mm well, that should give me
those 200 ng for the 10 kb , 1 kb and 0.5 kb bands. When I check
inserts from ligations by pcr, I get clear bands at 1 kb (for instance)
with easily more than 5 times the intensity of this 1 kb marker band
(and I can also see the remainders of the unused primers). So, this
should give me a "separation" of about 1 ug in a 4 mm well. Of course,
there is a slight smear, but nothing really disturbing your size
determination. I can send you some gel pictures, if you like ;-) I
am not sure about 2-3 ug per well, but 1 ug is no problem in a 0.8%
gel. Just try to load 1 ug of your plasmid on the gel and see whether
you can get the three conformation forms (though, in our hands we get
about 90% in ccc form).
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