[Histonet] the case of the disappearing dna

[Emily Sours]

This isn't really histology, more molecular biology, but I'm not aware of
any good molecular biology lists ^_^
I recently started making RNA probes again (after about a year of not doing
so).  When I linearize plasmid DNA (usually pBS, smaller than 4kb total)
and run it on a gel, the band is really really light.  I know the DNA I'm
starting with has a bright band when it's run uncut.
The protocol is a basic restriction enzyme digest for 2-3 hours, which I
then clean with a Qiagen kit.
I've tried running the DNA on a gel before I clean it, in case it's the
cleaning which is not working well, but it's still really light.
The weird thing is, if I use it to make an RNA probe, I get a pretty big
RNA pellet when I ethanol extract it.  It's also specific when it's used in
a slide in situ.  But again, I can't see any RNA on a gel AND the DNA band
is very light.
WTF is going on??
One thing I haven't checked is the temperature of the water bath--it's a
digital reading and over ten years old so it might be off.
I have tried different plasmids and different enzymes only to get the same
result.
When I used to make RNA probes, both the linearized DNA and DNA with RNA
probe were pretty bright.
Also, I tried different loading buffer and ethidium bromide for the gels.
Also also, I've run uncut DNA on the same gel and it was a very bright band.
Would it be worth it to take samples of the restriction digest before it's
incubated, and then at one hour intervals, to run on a gel with the uncut
DNA? I really have no solution for this, and using the RNA for an in situ
without seeing it on a gel makes me very nervous.
Sorry for the rambling nature of this email, it's Saturday and I keep
thinking random things I should mention about what I'm doing.

Emily



"You see a peanut, day's off to a good start; you witness some soil it's a
jamboree for Vince Noir."
--Howard Moon, in "Charlie", The Mighty Boosh