Tue Jan 23 13:30:38 EST 1996
In article <DLL7xL.Aro at info.swan.ac.uk>, Jim Harvey wrote:
> In article <kernan-1901961105160001 at powerlarry.mib.uga.edu>, kernan at uga.cc.uga.edu (Rich Kernan) says:
> >In article <4dlmpe$2os at gatekeeper.kcc.com>, "K.G. Korth" <kkorth at kcc.com> wrote:
> >> I have tried SYBR I Green in loading buffer (standard 6X TBE loading
> >> buffer with 1:10,000 SYBR I Green) as recommended by a newgroup poster
> >> about 6 months ago and found the migration to be significantly affected
> >> by the SGI, such that the bands that were supposed to line up, no longer
> >> did (including my ladder DNA). Same thing happened when adding the SGI
> >> to the agarose itself rather than the loading buffer. Though it did work
> >> as a post run stain, it is just too expensive to use that way and really
> >> gave us no better results than the EtBr (as a post run stain).
> >> I love the sensitivity of SGI and the very low background, but I could
> >> not tollerate the way it affected migration. We no longer use it for
> >> anything.
> >make size determination impossible. So I guess the prohibitively
> >expensive post staining is the only useful method. If anyone has found
> >a way to prestain with SGI then PLEASE post it.
> I must admit I didn't notice this at first, I was pre-staining genomic
> DNA preps with a 1:10000 dilution of SYBR green/loading buffer and it
> didn't appear to have any affect on their migration. I read you comments
> and tried some different concentrations 1:10-1:10000 on some PCR products
> and yep you guessed it bands all over the place. So fisrt of all sorry,
> if I misled anyone it was entirely unintentional.
> I'm still a bit annoyed about this though and consequently, I'm still
> trying to find ways to make its routine use financially possible.
> Which leads me onto my question of why the SYBR green reconstituted
> for poststaining'decays' so rapidly when made up according to the
> manufacturers instructions. Does anyone know?
> If we could find a way around that then our SYBR green troubles would
> be over.
> Now I wonder what would happen if you froze that.............?
I have found the same problem, but in my hands it isn't all that bad. I am
running 1.5% Synergel + 0.5% agarose gels in 1X TAE. Gels are run at very
low voltage 37V for 14 hours in the dark. My loading buffer is basically
the 6X Ficoll Loading buffer from Maniatis, but in 1X TAE, with the
addition of 200 ul of 0.5M EDTA (pH 8.0)/10 ml loading buffer, just before
use I add 0.5 ul of SyBr Green concentrate to 300 ul of LB (more or less as
suggested in this newsgroup). I add 3 ul of LB to approximately 10 ul of
sample (PCR products cut with 4-cutters), wait 10 minutes and run. I do
not feel confident scoring small size differences (say less than 20 bp in a
200 bp band), but have generally found the results to be pretty good (I
think I can always score a 50 bp difference even in large freagments).
Oddly when bands migrate at the "wrong" MW only a few samples are affected
for unknown reasons, I have the impression it is related to the amount of
DNA loaded. I guess my question is how serious is the effect in other's
hands?? There is enough of an effect in my hands that I would be
interested in post staining methods, (as post stained gels give generally
better resolution), but the cost right now is enough that I would be
inclined to make Southern blots and never stain the gel.
More information about the Methods