pTopo purification problems

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Fri Sep 11 02:33:04 EST 2009


I recently had a similar problem, where after plasmid prep/enzyme cleavage,
I would try to Gel Extract a fragment of the plasmid, only to find I had
none left at the end.

It turned out to be a bad Qiagen kit, it seems some of the buffers can cease
to work right if they are left to age too long, even if sealed. If the pH on
your buffers is wrong, you can expect to lose the DNA during preparation!
Which might necessitate either some titration or a new kit.

2009/9/10 Taliaferro, Dwayne (NIH/NIMH) [F] <taliaferrod from mail.nih.gov>

> Um...try it again?
>
> On a constructive note...
>
> What size is the gene?
> Did you -tail it?
> Did you try a control reaction?
> Perhaps it was a plasmid prep issue?
> Are you sure the colony you picked was bacteria vs fungus?
> Are you sure your AMP is working?
>
> On 9/10/09 3:54 PM, "Yoram Gerchman" <gerchman from research.haifa.ac.il>
> wrote:
>
>
>
> Greetings netters
> I have recently made an attempt to clone a short gene into pTopo (using
> Top10
> competent cells). i did get white colonies on LB-Amp-X-gal, and they grew
> nicely on liquid LB-Amp, but after plasmid purification I get no DNA (tried
> already 2 different kits). Any ideas as to how remediate this situation?
> Many thanks
> Yoram
>
>
>
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