Transfections using QIAGEN
QIAGEN at kaiwan.com
Fri Mar 24 13:44:34 EST 1995
Hi Nick and Jurgen,
In Article <nick.5.2F559DCF at lmb1.rug.ac.be> nick at lmb1.rug.ac.be
and in article <3jfav3$n2e at amcnix.amc.uva.nl> Jurgen
expressed concern over problems with low efficiencies of transient
transfections using QIAGEN columns.
Inconsistency in transfection may be related to purity of the DNA, which is
influenced by the amount of bacterial culture processed over the QIAGEN
column. If you are having purity problems, there are three points to
consider before performing a plasmid preparation. I call them the "3 C's"
culture volume, copy number of the plasmid, and capacity of the column.
Copy number: a neat rule of thumb to determine how much culture volume to
use to reach the desired plasmid yield is: 3-5 µg plasmid DNA / ml of LB
culture for high copy plasmids (pUC, Bluescript, etc) and 0.2-1 µg plasmid
DNA/ ml of LB culture for low copy number plasmids. This is how we
determine our recommended culture volumes (included with QIAGEN-tips) for
each column size (e.g. 100 ml h.c. plasmid yields approx. 500 µg).
Culture volume: When alkaline lysis conditions are optimal, P2 lyses all
the cells and P3 precipitates quite a bit of protein and all the gDNA. The
column procedure then removes the remaining protein, etc.
If too much culture is used, the efficiency of alkaline lysis is reduced
and the column is overloaded. All the protein and polysaccharides may not
be removed in the wash steps and find their way into your final eluate.
You should be able to avoid these contaminations by using the recommended
culture volumes or growing to the proper cell density (generally, 12-16 hrs
growth should be o.k.). Also, we do not recommend rich broth (TB, 2xYT) as
this increases your cell density 4-5 fold and the recommended volumes of
alkaline lysis buffers would not be optimal.
Capacity of column: the nomenclature of the column tells you the binding
capacity (e.g. QIAGEN-tip 500 binds 500 µg). I have talked to many users
who thought the number was indicating the culture volume to process.
Actually, using only 100 ml LB culture will yield approx. 500 µg of a high
copy number plasmid.
I hope this helps. There are other tips that we could give for isolation of
plasmid from low copy number plasmids if you are interested.
Temporary Email Address qiagen at kaiwan.com
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