"determination of cy3/cy5-dCTP incorporation"

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kscheirer Jun-20-01, 03:06 PM (PDT) "determination of cy3/cy5-dCTP incorporation" Does anyone know of a method to determine the incorporation of cy3/cy5-dCTP into a randomly primed genomic DNA probe? Thanks, Katie Scheirer Higgins Lab Univ. Alabama at Birmingham   Alert Edit | Reply | Reply With Quote | Top

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RE: determination of cy3/cy5-dCTP i..., Kazuo, Jun-22-01, (1) RE: determination of cy3/cy5-dCTP i..., martin overgaard, Jun-29-01, (2)

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Kazuo Jun-22-01, 01:46 PM (PDT) 1. "RE: determination of cy3/cy5-dCTP incorporation" LAST EDITED ON Jun-22-01 AT 01:47 PM (PDT) When using direct dye-incorporation labeling, monitoring the success of labeling reactions may be one of the critical success factors for high quality microarray data. You can find the detail protocols in the instruction manual of CyScribe (Amersham), You can get it on the website of Amersham. I tried, but it was not so practical. You need to know the yield of synthesized probes using radioactive tracer. You need also a microcuvette. Hope it helps. Kazuo   Remove | Alert Edit | Reply | Reply With Quote | Top

martin overgaard Jun-29-01, 06:01 AM (PDT) 2. "RE: determination of cy3/cy5-dCTP incorporation" Hi Katie In order to determine incorporation of Cy3/Cy5-dCTP I measure absorbance of the samples at their absorbance maxima that is 550 and 660nm for Cy3 and Cy5, respectively.Using water as blank and using the Beer-lambert law you can calculate the absolute molar amount incorporated in your samples. The extinctionkoefficients for Cy3 is 150.000 (cm-1, M-1) and for Cy5 250.000 (cm-1, M-1). Although it is possible to meassure the total amount of cDNA synthesized at A260, there might be some contribution in absorbance by the dyes. You can find correction factors at molecular probes, but not for Cy3 or Cy5. Perhaps you can find your own correction factors using free Cy-dye measurements at 550/650 and 260nm. Good luck Martin Overgaard Microbiology group SDU, Odense, Denmark   Remove | Alert Edit | Reply | Reply With Quote | Top

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