"2-step Cy-dye labelling, differences in incorporation"

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pdetter Aug-07-00, 02:02 PM (PDT) "2-step Cy-dye labelling, differences in incorporation" Hi, I'm wondering if anyone has had the experience when using the two-step (aminoallyldUTP) labelling process to label cDNA where you get much less signal from the Cy5 dye even when using the same amount of mRNA for both labelling reactions? I'm fairly new at this and have only done two hybridizations so far, but both times the Cy5 signal has been much lower than the Cy3 signal. I have heard that generally, if anything, Cy3 incorporation and fluorescence is lower when labelling with Cy-conjugated dUTP in the one step process. Is monofunctional Cy5 coupled less efficiently to incorporated aminoallyl-dUTP to anyone's knowledge? I can't seem to find any references that address this issue. Thanks for your help if anyone can provide some insight. Paul Etter MCB Dept. University of Arizona   Alert Edit | Reply | Reply With Quote | Top

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RE: 2-step Cy-dye labelling, differ..., aestep, Sep-09-00, (1) RE: 2-step Cy-dye labelling, differ..., BPostier, Mar-30-01, (4) RE: 2-step Cy-dye labelling, differ..., DavidShafer, Jan-04-01, (2) RE: 2-step Cy-dye labelling, differ..., stephaniesommer, Jan-25-01, (3) RE: 2-step Cy-dye labelling, differ..., Xiuling Wang, May-23-01, (5) RE: 2-step Cy-dye labelling, differ..., Xiuling Wang, May-23-01, (6)

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aestep Sep-09-00, 12:48 PM (PDT) 1. "RE: 2-step Cy-dye labelling, differences in incorporation" You have company. We have been conducting hybs, both direct incorporation and amino allyl, for some time now. Cy3 always binds better and we have no idea why. Anne   Remove | Alert Edit | Reply | Reply With Quote | Top

BPostier Mar-30-01, 01:58 PM (PDT) 4. "RE: 2-step Cy-dye labelling, differences in incorporation" We have been doing the same, with again a lower signal in the cy 5, but i don't beleive it is due to a lower binding. I beleive that cy 5 doesn't fluoresce as brightly as cy 3 due to the structure of the molecules, but I could be wrong.   Remove | Alert Edit | Reply | Reply With Quote | Top

DavidShafer Jan-04-01, 01:23 PM (PDT) 2. "RE: 2-step Cy-dye labelling, differences in incorporation" Hi, I am looking for the protocol for aminoallyl-dUTP labeling. It used to be in the PBrown list. Missing now. Any help? David   Remove | Alert Edit | Reply | Reply With Quote | Top

stephaniesommer Jan-25-01, 01:02 PM (PDT) 3. "RE: 2-step Cy-dye labelling, differences in incorporation" Here's another one joining the club. If anyone finds out anything useful, please let me know. Good luck! Stephanie   Remove | Alert Edit | Reply | Reply With Quote | Top

Xiuling Wang May-23-01, 01:47 PM (PDT) 5. "RE: 2-step Cy-dye labelling, differences in incorporation" Dear Paul: I am using RT-probes with labelling Cy3 and Cy5. We have stranger Cy5 signal than that in Cy3. Have you compared these two labelling methods? Do you have any ideas about their differences of labelling? Yours Truly Xiuling Wang   Remove | Alert Edit | Reply | Reply With Quote | Top

Xiuling Wang May-23-01, 01:48 PM (PDT) 6. "RE: 2-step Cy-dye labelling, differences in incorporation" Dear Paul: I am using RT-probes with labelling Cy3 and Cy5. We have stronger Cy5 signal than that in Cy3. Have you compared these two labelling methods? Do you have any ideas about their differences of labelling? Yours Truly Xiuling Wang   Remove | Alert Edit | Reply | Reply With Quote | Top

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