Re: [DIYbio] Re: trouble loading onto agarose gel

On Sun, Mar 24, 2013 at 1:25 PM, Jeswin <phillyj101@gmail.com> wrote:
> On Wed, Mar 20, 2013 at 9:16 PM, shamrock <thmsburkett@gmail.com> wrote:
>> I hate it when that happens! For the sample to fall to the bottom of the
>> well it as to be denser then the surrounding buffer. Nathans right that
>> loading dyes typically have either glycerol or sucrose to increase the
>
> I did a test using just the Roche buffer, water, and increasing
> volumes of loading dye. I couldn't reproduce the effect.
>
> So i went about cutting, first with the Roche enzymes.I did
> PCR-purification after cutting was over. Then I did the second cut
> with the NEB enzyme. I tried to load it on the gel and ran into the
> same problem. But this time I used an undiluted Loading buffer (from
> the Qiagen Kits). It helped a bit but the mix didn't spread out as
> usual. Like there was some at the ends and a little bit fell on the
> center.
>
>> density, and yes ethanol contamination by decreasing the density can cause
>> samples to float away. Does Roche supply the composition of their buffer? It
>> could be that an organic would decrease density enough to not allow it to
>> enter the gel but this seems like a pretty big oversight on the part of
>> Roche.
>
> Roche Buffer M (NheI) : Tris-HCl 10mM, MgCl2 10mM, NaCl 50mM, DTE 1mM
>
> For reference: NEB Buffer 2 (NheI): 50 mM NaCl, 10 mM Tris-HCl, 10 mM
> MgCl, 1 mM dithiothreitol
>

DTT vs DTE are just diastereomers, I can't find any data on
differences in viscosity or density but I think it would be small,
especially since their concentration is pretty low (1 / 71mM)

You didn't mention what the elution buffer was, or what the ratios of
loading dye to sample was for each.

Why aren't you doing an ethanol precip before cutting anyway?


--
-Nathan

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