[Zbrafish] DNA preps for injections

James Lister via zbrafish%40net.bio.net (by jalister from vcu.edu)
Wed Dec 17 08:24:38 EST 2008


Hi Michelle,

Actually, we use Qiagen spinprep DNA (or more recently the equivalent kit
from Fermentas) for pretty much everything with no problem.  True, we might
get better results with cleaner preps of DNA but it hasn't been an area
we've felt a need to optimize.  We routinely are injecting in the range of
10-50 pg per embryo, and certainly see toxicity at the upper range of that,
but for the most part get sufficient survival/normal development to work for
us (experiments perhaps similar to what you are talking about -- screening
potential promoters/enhancers with GFP, etc.?)  I assume you've already
tried titrating down the amounts you are injecting to be sure it's a DNA
quality (vs quantity, or other parameter) problem?

As for BACs, there may be something better on the market for this now, but
when I last did this a few years ago I basically used the Qiagen midi kit
with a 100 ml culture to get a miniprep-sized yield (10-20 micrograms).  The
only adjustment to the protocol that was made was to pre-heat the elution
buffer to 65C.  For quick screening one can do a 2-5 ml culture and use the
Qiagen reagents for the early steps, but instead of the column step do a
phenol/chloroform extraction followed by an isopropanol precipitation (there
used to be a protocol for this on a web site somewhere but I can't seem to
find it now).  As you might expect this is going to be dirtier than a column
purification but the yield is much better.  And my experience with BACs is
that you don't need to inject very much even though they're much larger,
because they will express much more strongly than a plasmid that has a
relatively short promoter.

Hope this helps (I'm curious to hear what others do as well),
Jim
 

> -----Original Message-----
> From: zbrafish-bounces from oat.bio.indiana.edu 
> [mailto:zbrafish-bounces from oat.bio.indiana.edu] On Behalf Of 
> Michelle Emond
> Sent: Tuesday, December 16, 2008 6:02 PM
> To: zbrafish from magpie.bio.indiana.edu
> Subject: [Zbrafish] DNA preps for injections
> 
> Hi,
> 
> We've been trying to find a reliable kit for cleaning up DNA for 
> injections.  Qiagen miniprep DNA does not seem to be clean enough to 
> inject straight off the column, as the embryo survival rate is very 
> low.  So, we've been running the DNA over one of their PCR 
> purification 
> columns to further "clean" the DNA, and that seems to result 
> in higher 
> survival rates.  However, we'd like to streamline our methods 
> to cut costs.
> 
> We'd love to hear what other labs do for DNA preps (plasmids 
> and BACs), 
> both for larger preps of known constructs (midi or maxi), and for 
> miniprep quantities for screening potential constructs.
> 
> Thanks very much,
> Michelle Emond, Ph.D.
> email:  emond.4 from osu.edu
> Center for Molecular Neurobiology
> Ohio State University
> 
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