How to measure Cy5-primer concentration ??

Jose de las Heras josenet at tiscali.co.uk
Fri Oct 6 15:54:18 EST 2006


"DK" <dk at no.email.thankstospam.net> wrote in message 
news:cHtVg.1209$iP5.1105 at newsfe07.lga...
> In article <4omk7vFf8rjcU1 at individual.net>, "Peter Ellis" 
> <pjie2 at cam.ac.uk> wrote:
>>DK wrote:
>>> <pjie2 at cam.ac.uk> wrote:
>>>> DK wrote:
>>>>>
>>>>> Cy5 almost certainly absorbs srtongly at 260-280 (two indole rings).
>>>>
>>>> It doesn't.
>>>
>>> Sure it does. What is there in Cy5 structure that indoles suddenly
>>> would
>>> stop absorbing ultraviolet? Nothing. Sorry, I don't believe it.
>>
>>Go and try it then.
>
> What is there to try? it does absorb - see Duncan's post.
>
>>In the context of fluorescently labelled DNA, Cy5
>>fluorescence at ~260 is negligible.  Every group working with labelled DNA
>>knows this (or should).  It's how you quantitate the efficiency of your
>>labelling - you measure the DNA concentration by looking at 260, the Cy5 
>>(or
>>Cy3) concentration by looking at their absorption maxima, etc.
>>
>>The signals do not interfere.
>
> They do, you just don't care about ~ 5% error you willingly introduce.
>
> DK

I guess that most of these deal with cDNA labelling and the like: i.e: 
relatively large molecules (compared to primers)  with relatively low 
density of Cy5 incorporated, so that the effect is not so noticeable.

It's been an enlightening thread, as I also just *assumed* Cy5 and Cy3 
contribution would not matter ever. And yes, I only deal with labelled cDNA 
molecules.

Thanks to all!

Jose 




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