"Black holes and Label with ARES Kit"

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bobice Dec-07-01, 01:46 AM (PDT) "Black holes and Label with ARES Kit" I've performed a few hybridizations these days, but for one channel, all of slides appeared black holes where DNA printed, while background was very high; and no signals in the other channel at all. The probe was made by incorparation of aa-dUTP, and then coupled with Dyes with ARES Kit from Molecular Probe. Since the dyes' emission and excition wavelength is similar with Cy3 and Cy5, I used corresponding laser and filter as Cy3 and Cy5 used. I can't understand what happened for the black hole and only signal exist in one channel! Anyone met with the black holes before? And any one used ARES Kit for microarray? Any information was welcomed.   Alert Edit | Reply | Reply With Quote | Top

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RE: Black holes and Label with ARES..., cdefilippo, Dec-19-01, (1) RE: Black holes and Label with ARES..., cdefilippo, Dec-19-01, (2) RE: Black holes and Label with ARES..., bobice, Dec-27-01, (3) RE: Black holes and Label with ARES..., gerti, May-01-02, (4)

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cdefilippo Dec-19-01, 11:04 AM (PDT) 1. "RE: Black holes and Label with ARES Kit" Hi, the black holes are due to the fact that the DNA printed on your glass slides protects the spot region from the fluorescence of the labelling mix. For this reason the fluorescence values of the spots are less than the background. I never used ARES Kit from Molecular Probe, but probably you have not incorporation of dyes in your samples. Try to use the De Risi protocol (amino-allyl-protocol) at the site: http://derisilab.ucsf.edu/ Good luck Carlotta   Remove | Alert Edit | Reply | Reply With Quote | Top

cdefilippo Dec-19-01, 11:08 AM (PDT) 2. "RE: Black holes and Label with ARES Kit" Hi, the black holes are due to the fact that the DNA printed on your glass slides protects the spot region from the fluorescence of the labelling mix. For this reason the fluorescence values of the spots are less than the background. I never used ARES Kit from Molecular Probe, but probably you have not incorporation of dyes in yoour samples. Try to use the De Risi protocol (amino-allyl-protocol) at the site: http://derisilab.ucsf.edu/ Good luck Carlotta   Remove | Alert Edit | Reply | Reply With Quote | Top

bobice Dec-27-01, 10:44 PM (PDT) 3. "RE: Black holes and Label with ARES Kit" LAST EDITED ON Dec-27-01 AT 10:48 PM (PDT) Thank you, Carlotta. I tried to label with Cy3-dUTP, and the probe works well - black well disappeared. But for the probe I hybridized last time, I don't think dyes didn't incorporate into cDNA. Before I hybridized my probe on the slide (after purifying probe using Microcon-30 and Ethanol precipitation), I spot a serially diluted (far to 1:10,000) samples onto a coated slide, then UV-crossed and whashed with 0.05 SSC, then scanned. The signal was there even diluted 10,000 times! Do you think it's a good method to check probe quality by this way? guanbin   Remove | Alert Edit | Reply | Reply With Quote | Top

gerti May-01-02, 09:22 AM (PDT) 4. "RE: Black holes and Label with ARES Kit" I have used the Alexa dyes for a while and they worked fine until I ordered a new lot (it took me forever the figure that out) it appears to me that the lots starting with 72 are bad. When I use them I get the black holes too. I still had a more than 1 year old sample labeled with an older lot and guess what that looks a lot better. My suggesting go complain with mol probes too. Normaly their stuff is pretty good but not this lot. gerti   Remove | Alert Edit | Reply | Reply With Quote | Top

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