Hello all,I had no problems with Cy5 signals for months. Since about two months however, I don't get any signal in the Cy5 channel. I've done all the following basic troubleshooting :
(1) Tried different RNA samples, including dye swaps. Great Cy3 signal all the time (high signal, no background), but no Cy5 signal AT ALL. Just background. So it's not the RNA or the enzyme (Superscript RT III) or the solutions.
(2) Tried different batches of Amersham Cy5. No signal whatsoever.
(3) Checked incorporation of the dyes with ODs 650 and 550. Both look reasonable. So the Cy5 gets incoroporated and is being degraded somewhere during hybridization, washing or scanning.
After extensively searching the web, people suggested it might be ozon in the summer and suggested adding DTT.
(4) I added 1 mM DTT to all solutions. They are also filtered and autoclaved. No Cy5 signal, Cy3 good as always.
Some posts suggested it might be the milliQ water with old filters.
(5) Tried double-distilled water and commercial VWR deionized water. Still no Cy5 signal at all.
Called Amersham tech-support : not of any help.
I am running out of ideas. Do any of you experience or have experienced problems with Cy5 degradation (no signal) when everything seems OK (incoroporation, enzymes, RNA ...)
Any help would be greatly appreciated,
Stephan
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Stephan EBERHARD
Grossman lab'
Carnegie Institution of Washington
Department of Plant Biology
260, Panama Street
Stanford, CA 94305
United States of America
Tel. : (650) 325 1521 x 284
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RE: Problems with Cy5,
JeremyH, Jul-30-04, (1)