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

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

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

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