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High Fluorescence using SYTO9 Dye

High Fluorescence using SYTO9 Dye - DNA Techniques

High Fluorescence using SYTO9 Dye - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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Old 08-30-2011, 06:51 PM
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Talking High Fluorescence using SYTO9 Dye



Hi Everyone,

I am having trouble with very high background fluorescence when using intercalating dyes. I am designing a high resolution melt experiment and I have been using the recomended concentration of MeltDoctor (SYTO9 green) at 1 uL/reaction with a reaction volume of 25 uL. The cycle threshold often times, when autoanalyzed will come up at 60,000-1,000,000. Is this normal? I am used to using a taq-man probe assay where the cycle threshold will be around... 0.2! I have not attempted to decrease dye concentration yet because I have not optimized other important components (primers and MgCl first).

Any information would be great because I am not having any luck finding info about this on the web.

Brittany


Last edited by brittanycronk; 08-30-2011 at 06:52 PM.
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