Our lab is actually making the change from ethidium bromide to GelRed
right now. We opted for GelRed over GelGreen because it works with our
UV transilluminator set-up without any modifications to protocols.
Real time PCR folks were too particular to switch from SYBR.
We had problems with smearing on our first test run following the
manufacturer's instructions, so we ran a test. Our best result was a
1:50,000 GelRed:Gel concentration. A neighboring lab uses 1:20,000.
Theory is that since the GelRed is so sensitive, the concentration
should be lower. Additionally, due to increased sensitivity we can use
our ladder at 1/6th concentration. Theoretically, lower DNA load
requirements could reduce PCR cycles and related reagent consumption
and boost experimental sensitivity, albeit by <6x.
I believe much of this should translate to GelGreen, but I would
encourage you to run a few gels of varying concentrations and ladder
load to see what works best. Type of gel you are running could be a
factor as well. We use agarose.
My only concern with these replacements is the sole source nature. I
could easily see a marketing strategy with low entry prices for 2 more
years giving way to price inflation once adoption is widespread. Non-
competitive pricing is not unique to gel staining, and I believe
largely responsible for the cost of Bio.
On Feb 6, 8:01 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
> From the Biotium (the compkany that makes gelgreen and gelred) the
> stain works on both ssDNA and dsDNA, so its not just doing
> intercalation.
>
> From the same document it seems like the company doesn't even really
> know... or they are being a bit obscure
> "What is the binding mechanism
> of GelRed/GelGreen?"
> "GelRed and GelGreen most likely bind by a combination
> of intercalation and electrostatic interaction."
>
> Also:
>
> "In which direction does
> GelRed/GelGreen migrate?"
>
> "GelRed and GelGreen do not migrate through the gel as
> easily as EB. It is not necessary to add additional dye to
> the running buffer, and the gel will be stained more
> homogenously than that of EtB"
>
> (where EB and EtB are assumed to be Ethidium Bromide, which has a +
> charge and migrates in the opposite direction of DNA)
>
> http://www.biotium.com/product/product_info/other/GelRed%20and%20GelG...
>
> I personally think post-staining is the best method, as you KNOW it
> won't affect the migration of the DNA bands at all. Post-staining
> solution keeps working well for at least 1.5 - 2 months before you
> need to add a little more stock solution.
>
>
>
>
>
>
>
>
>
> On Mon, Feb 6, 2012 at 2:19 PM, cameron <seluronk...@gmail.com> wrote:
> > There's a small group of us that are working with GelGreen at
> > Biocurious in an effort to find the most efficient and cost-effective
> > way to use it. We've tried pre-staining (in gel) and post-staining
> > (in buffer) and both work well. We were wondering if there is any way
> > to combine the stain with the DNA prior to or at the time of -
> > insertion into the well of the gel. We haven't been able to find any
> > information about how it actually stains. We ran a quick test last
> > night and found that it does migrate with the DNA but not cleanly.
> > Does this make any sense or are we wasting our time? Has anyone
> > experimented with this or know how Gelgreen stains the DNA?
> > Thanks,
> > Cameron
>
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> --
> Nathan McCorkle
> Rochester Institute of Technology
> College of Science, Biotechnology/Bioinformatics
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