"Cy5 signal disappeared"

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simonkayin Sep-04-01, 08:14 AM (PDT) "Cy5 signal disappeared" Hi, I am a microarray beginner and I absolutely needed help! I have problem working with Cy5 dye. I use indirect incorporation protocol. After labelling and purification with Qiaquick PCR purification kid, I measure the abs at 550 and 650 nm. Both cy3 and cy5 labelled cDNA give me similar incorpaoration ratio. Then I dried down the labelled cDNA with SpeedVac and resuspend in 50% formamide hybridization buffer and hybridize at 42o C overnight. However, after washing, all the Cy5 signal disapeared and look like I haven't add Cy5 labelled cDNA(which I am sure I added). The whole slide only give strong green colour (Cy3 signal). I use axon 4000A and 4000B scanner. Can anyone give me suggestion what happened? What can I do about it? Thanks.   Alert Edit | Reply | Reply With Quote | Top

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RE: Cy5 signal disappeared, jkday, Dec-05-01, (1) RE: Cy5 signal disappeared, danielmaclean, Dec-07-01, (2)

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jkday Dec-05-01, 12:25 PM (PDT) 1. "RE: Cy5 signal disappeared" Did you ever resolve your problem with the Cy5 signal disappearing? We have been having similar results using the Qiaquick kit. Thanks >Hi, > >I am a microarray beginner and >I absolutely needed help! > >I have problem working with Cy5 >dye. I use indirect incorporation >protocol. After labelling and purification >with Qiaquick PCR purification kid, >I measure the abs at >550 and 650 nm. >Both cy3 and cy5 labelled >cDNA give me similar incorpaoration >ratio. Then I dried down >the labelled cDNA with SpeedVac >and resuspend in 50% formamide >hybridization buffer and hybridize at >42o C overnight. However, after >washing, all the Cy5 signal >disapeared and look like I >haven't add Cy5 labelled cDNA(which >I am sure I added). >The whole slide only give >strong green colour (Cy3 signal). >I use axon 4000A and >4000B scanner. > >Can anyone give me suggestion what >happened? What can I do >about it? > >Thanks.   Remove | Alert Edit | Reply | Reply With Quote | Top

danielmaclean Dec-07-01, 10:30 AM (PDT) 2. "RE: Cy5 signal disappeared" I have had a similar problem. After purification with an amersham autoseq g-50 kit the cy-dye could be seen to flouresce when cDNA was run on a gel and visualised using a Typhoon imager but not using the spec. Could it be that specs are not sensitive enough to detect the cy-dye when diluted? Any ideas any one? Thanks   Remove | Alert Edit | Reply | Reply With Quote | Top

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