DNA dyes

HENRY DAUM henry_daum at FMC.COM
Thu Aug 17 11:01:56 EST 1995


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Joanne,

I received your message and associated reply.

We have just finished compiling tips for optimization of the use of SYBR Green I
dye, learned in our research labs at FMC BioProducts, and from our customers.
Please see the enclosure.

We recently did the following experiment, which you may also be interested in:
        Electrophorese DNA in SeaPlaque GTG agarose
                (Use a TAE buffer containing an EDTA concentration of 0.1 mM)
        Visualize using SYBR Green I, 300 nm transillumination
                (Keep UV exposure to less than 60 seconds)
        Excise the band containing the DNA (ca. 2kb)
        Melt the gel slice and add a fraction to appropriately linearized pUC 18
        Ligate in the presence of the agarose
                (A weak gel WILL reform during ligation)
        Remelt the ligation mixture, dilute, and use for transformation.
        
The experiment worked well. Protocol followed was essentially what we describe 
in the SeaPlaque GTG product literature, and is in the back of our 1995 catalog.

Also, for optimal DNA visualization in your agarose gels, be certain that the 
gel thickness is 4 mm or less. This is VERY important.

Hank Daum, III
Technical Product Specialist
FMC BioProducts

______________________________ Reply Separator _________________________________
Subject: Re: DNA dyes
Author:  Jfloring at aol.com at Internet
Date:    8/16/95 3:05 PM


Brian D. Lanoil (lanoilb at ava.bcc.orst.edu) wrote:
>I did try it a while back, and I found little difference between SYBR 
>Green and EtBr.  In fact, there was a disadvantage that I saw--the 
>exposure times you need to use for imaging the SYBR Green-stained gel was 
>10-fold longer than for EtBr.  It is true that there is no background at 
>all, but the signal was so faint that I couldn't see it with my eye 
>alone, even at high DNA concentrations.  
     
I've fooled around with SYBR green I, and found it to be always BRIGHTER than 
EtBr.  However, after one use, the solutions did not work very well for us, 
and perhaps that's what happened to you.   Also- the filter on the camera 
which is used for EtBr is not right for SYBR green, and that may also be 
increasing the exposure time.  Try using a fresh solution, if you haven't
 yet.
     
>The main advantages I saw to SYBR green is that it does not appear to 
>interfere with restriction digestion, unlike EtBr (rest omitted)
     
I hadn't realized this.  Is this part of the official documentation, or an 
observation you made?  
If this is indeed true, I'll use SYBR green in spite of the cost. 
Cheers, 
Jeanne Loring
     
     
     
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From: Jfloring at aol.com
Subject: Re: DNA dyes
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////////////////////////////////////////////////////////////////////////
////////////////////////////////////////////////////////////////////////
////////////////////////////////////////////////////////////////////////
/////////////////////////////////////////////////////////////////////w==
--IMA.Boundary.490576808--



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