UV and cloning, ETB (was: pcr prob)
(by maximilianh from gmail.com)
Thu Feb 28 04:44:37 EST 2008
I don't have a publication for it, but yesterday I've stumbled over
this post. The author is describing that 1 minute of UV destroys most
of your clones. (well, I don't find it too difficult to keep the times
down to 10 seconds, it's still nice to see that someone tried this in
a more systematic way)
BTW there is also a post about ethidium bromide toxicitiy:
On 28/02/2008, DK <dk from no.email.thankstospam.net> wrote:
> In article <4fa05c91-0b6e-471b-a8f7-62ad3856d3c7 from u72g2000hsf.googlegroups.com>, Tom Landers <galaxyglue from gmail.com>
> >After reading this *very* embarrassing pissing contest, i'm amused to
> >point out that none of the participants examined the assumption that
> >EtBr was the stain used.
> >I always see primer bands in gels stained with "Sybr Safe".
> But are these primers? :-)
> My understanding is that Sybr Safe, just like EthBr, does NOT
> stain ssDNA!
> >In fact, presence or absence of a primer band in a lane with no
> >amplification band helps diagnose whether the failure is of the
> >amplicon, or of the liquid handling robot (i.e. no primers added to
> >that reaction).
> >You guys -- assuming that such pomposity and rudeness is the sole
> >province of male scientists -- are a real testament to the profession.
> >P.S. - You're still using ethidium stain? Really??
> Really! And, for as long as we are not in a position of not
> knowing where to spend money, I see no absolutely reason to
> change that.
> 1. "Safe" part does not concern me in the least. EthBr is already
> perfectly safe the way it is used.
> 2. Sybr Safe is something like 20X more expensive, isn't it?
> 3. The only clear advantage Sybr Safe is lack of UV damage to
> the preparative DNA purifications. However, it seems that to
> realize that advantage one has to buy a new/special
> transilluminator. Not cheap! Plus, even with the UV damage,
> using our home-made electrocompetent cells always gives me
> 100-1000X more clones than I could ever possibly screen -
> so that advantage is lost on me as far as what we routinely do
> 4. Correct me if I am wrong but isn't Sybr Safe slightly less
> sensitive than EthBr? At least that's couple people who actually use
> it told me.
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