I am experiencing a green haze over the entire chip. I have tried to
eliminate it by ensuring the glassware is clean, no latex gloves come in
contact, no contamination that I am aware of.
Has anyone run into this?
Appreciate your help.
"sklwater" <[Only registered users see links. ]> wrote in message
news:m9PTg.1381$[Only registered users see links. ].pas.earthl ink.net...
I've had that a couple of times.
I use a waterbath to incubate the slides during hybridisation. Once I had
some anti-fungal stuff on the waterbath, it has a blue dye in it... although
no water gets into the microarray chamber it was enough to get veyr high
uniform background on all my slides on the Cy3 channel. So I never add
anything to the water, just clean it regularly.
Do you use the new Qiagen PCR purification kit with the yellow dye on the
binding (PB) buffer to clean up your labelling reaction?
I did. It's terrible.
I use a Nanodrop to check the labelling and you can see a very high
background in the spectrum, after clean up, when using the yellow PB buffer.
Even after repurifying the labelling I could still see a higher background
than usual... I am sure that without the extra purification teh slide would
be nearly unuseable.
So, if you use the yellow PB buffer... don't. I contacted Qiagen about it
and they sent me a large bottle of dye-free PB buffer free of charge the
SDS also fluoresces nicely on the Cy3 channel, so if the slides haven't been
washed & rinsed well you may get some...
I don't take special care with the glassware, not anymore than I'd take when
doing chromosome FISH, for instance. I clean all the glassware I use with
running water and rinse in distilled water... I regularly clean it well
using regular washing liquid, and then make sure it's all well rinsed away
by running water on it for a while. I use gloves (wash my hands on them
once, to remove dust, and that's it). I once made the mistake of cleaning
the back of a slide with a tissue dipped in ethanol... (this was using a
white light scanner that focuses on the spots from the back, through the
slide)... Not good... some stuff either on the ethanol or more likely in the
tissue was smeared all over the slide, fluorescing brightly.
If the background is something new that you didn't experience before, you
should check what it is that you're doing differently.
I'm sorry I cannot offer any certain answers.
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