UV and cloning, ETB (was: pcr prob)

Maximilian Haeussler via methods%40net.bio.net (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)
http://bitesizebio.com/2008/02/26/turn-away-from-the-uv-light/

BTW there is also a post about ethidium bromide toxicitiy:
http://bitesizebio.com/2007/09/26/ethidium-bromide-a-reality-check/

max

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>
>  wrote:
>
> >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
>  goes.
>
>  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.
>
>
>  DK
>
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-- 
Maximilian Haeussler,
tel: +33 6 12 82 76 16



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