pcr prob

Tom Landers via methods%40net.bio.net (by galaxyglue from gmail.com)
Wed Feb 27 21:49:15 EST 2008


> Pffft! Another one comes tumbling in through the door.
Ashamed to say i couldn't resist. Honoring primate ancestry, perhaps.

On Feb 27, 6:57 pm, d... from no.email.thankstospam.net (DK) wrote:
> In article <4fa05c91-0b6e-471b-a8f7-62ad3856d... from u72g2000hsf.googlegroups.com>, Tom Landers <galaxyg... 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? :-)

Yes  :p

>
> My understanding is that Sybr Safe, just like EthBr, does NOT
> stain ssDNA!
>

This was the reason i chimed in on this thread - the original poster
never said what stain was used. The fight assumed ethidium though.
Manufacturer's web site doesn't say anywhere that it doesn't stain
ssDNA.
Supposedly the Sybr dyes interact with the sugar-phosphate backbone,
unlike ethidum's base intercalating (thus its specificity for dsDNA).

> >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??
>

Snotty remarks, somebody punch me

> 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.

Of course. But there is a disposal cost associated with it. My gels
are considered "nonregulated waste" which makes the safety office
happy.

>
> 2. Sybr Safe is something like 20X more expensive, isn't it?
>

Yes.
http://probes.invitrogen.com/products/sybrsafe/

> 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!

Their blue light transilluminator is the same idea as the Dark Reader.
These work with ethidium too.

> 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.

Close enough to the same, in my experience. Manufacturer claims so
too.

>
> DK



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