trouble loading onto agarose gel

[Jeswin]

I did a restriction enzyme cut of a pcr product and wanted to run the
gel and purify it. After adding the loading dye, I proceeded to put
the mix into the well. Unlike other times that I loaded, the mix just
seemed to float away. Only by being extremely careful and extremely
slow was I able to load a good amount. Usually I load 25uL in the well
but this time, I had to load all 45 uL and I think most of it didn't
get in the well.

The last time this happened, I was told it was ethanol contamination
from purification so I took utmost care to reduce this. I was doing
double digests so I did PCR clean up after the first cut. The only
thing different with this particular cutting is that I used Roche NheI
and SuRE cut Buffer M. The NheI had an odor to it; an organic smell
like DMSO. I have not had problems with NEB RE.

Any ideas?

Thanks

[Nathan McCorkle]

Use thicker loading dye (more viscous)

http://openwetware.org/wiki/Agarose_gel_loading_buffer

sucrose (table sugar) and glycerol are common density agents

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[shamrock]

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 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.

[Jeswin]

On Wed, Mar 20, 2013 at 9:16 PM, shamrock <thmsb...@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

I also cut with SalI in the second cut but I can't be sure that is a
cause. I have used SalI without any problems

[Dakota Hamill]

these are easy to make and work http://openwetware.org/wiki/Agarose_gel_loading_dye

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[Nathan McCorkle]

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