Well, one of the nice things about having a 4 cartridge sequencer is that
you load it up and literally rotate through samples and cartridges. As
luck would have it, 3 out of 4 bands had high background from the 1 blot in
question, and those 3 bands were all run using cartridge C on the Procise.
When I saw the high background the third time, in spite of having washed
the bands in 50% methanol, followed by water, followed by air drying the
bands (in response to Bente Lou Isbye's question of yesterday. I find that
using the argon dry device on the Procise often blows the bands right out
of the cartridge so I no longer risk it.), I KNEW there had to be some
other explanation. To my surprise, I discovered that I had used the same
cartridge.
I ran several cycles using Cartridge C and no sample, only the Zytex
seal... You guessed it- huge background. I then sonicated the cartridge
(upper and lower pieces) in 50% nitric acid, rinsed well with water, and
reran the Cartridge blank test and still had unacceptably high background,
though it was reduced. I will retreat with Nitric acid and test again.
(It had to be the cartridge since the background did reduce and since the
other cartridges were tested and did not have the contamination problem.)
On looking back at the history of my sequencer runs, the sample used in
Cartridge C immediately before the first band from this blot had expected
levels of background, so the contaminant some how had to be associated with
that first band.
I think I have had a cartridge contamination problem maybe twice in the
appx 14 years I have been running a sequencer. For that reason, it is not
always the first thing I would think of. An obvious answer would be to
periodically test empty cartridges for their background levels, but I am
ususally trying to keep my backlog down and my head above water.
Best regards,
Matt Williamson