Micro Array

Jose de las Heras josenet at tiscali.co.uk
Sun Oct 1 08:35:54 EST 2006


"sklwater" <sklwater at earthlink.net> wrote in message 
news:m9PTg.1381$Lv3.1037 at newsread1.news.pas.earthlink.net...
>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.
>
> Alan

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 
next day.

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.

Good luck!

Jose
-- 
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