Transgenic mice vector construct
(by R.Jayakumar from roswellpark.org)
Tue Dec 4 15:09:19 EST 2007
I would not do PCR of a construct to use for microinjection, unless you
are sure the PCR did not introduce any artifactual mutations in the
construct especially the promoter and cDNA sequence. Alternatively, you
could point mutate the construct at the location where you want to
linearise to introduce a restriction site of your interest and then
linearise it. Hope that helps.
From: methods-bounces from oat.bio.indiana.edu
[mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Pow Joshi
Sent: Tuesday, December 04, 2007 2:52 PM
Cc: methods from magpie.bio.indiana.edu
Subject: Re: Transgenic mice vector construct
On 04/12/2007, rosaak <rosaak from gmail.com> wrote:
> Hi all,
> I have constructed a vector consting cDNA for making transgenic mice.
> The backbone is pCI contining CMV promoter.For microinjections i need
> to linearise the vector., and there are no suitable sites for it.
> My doubt is that can i use a set of primers to pcr amplify
> CMVpromoter- cDNA-polyA region from the construct and use it for
> [i'll be using a highfidility enzyme for pcr.]
> Is it techically ok to do this.If so how will i purfy it ?
well, I cannot say anything about the pcr,
for purification, I use the Qiagen kit, and it works quite well, at
least for my purposes.... but then Iuse zebrafish eggs.
You could also use the standard phenol chloroform extraction foillowed
ny isopropanol/ ethanol precipitation.
Hope that helps
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