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Weirdest thing with DNA dye and agarose gel


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4 replies to this topic

#1 Ahrenhase

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Posted 11 December 2013 - 12:02 PM

So I recently joined a new lab.  When I make gels, I microwave the agarose until there are no 'beads', immediately add 1/10,000 SYBR SAFE stain, let congeal, and then run the gel.  However, every time I do this, and image the gel under a UV gel box, there's hardly any fluorescence.  So what I have to do is post stain with SYBR (shake for 15 mins room temp) and it looks fine.  I've always made gels the way I'm doing it now and it's worked fine.  Any idea why I'm getting this weird result?

 

EDIT: I let gels congeal in the dark (usually within a drawer). 


Edited by Ahrenhase, 11 December 2013 - 12:04 PM.


#2 jerryshelly1

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Posted 11 December 2013 - 02:46 PM

Did someone photobleach the safe stain?



#3 mdfenko

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Posted 12 December 2013 - 05:08 AM

a couple of thoughts...

 

the sybr may be migrating out of the gel (like etbr)

 

and/or

 

the gel solution may be too hot when you add the sybr (we allow the agarose to cool some before adding etbr and pouring)


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#4 Ameya P

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Posted 13 December 2013 - 12:40 AM

Ahrenhase, 

 

Are the both the safe stains that you are using the same? or are they different? Any body else in the lab having similar problems? Or are you working all alone? 


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#5 phage434

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Posted 13 December 2013 - 04:16 AM

Try adding a small amount of sybr safe to the positive buffer pool. This will replace the sybr which is migrating toward the negative terminal.






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