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Smeared DNA ladder


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#1 hjb60

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Posted 13 November 2013 - 04:01 AM

Hi!

 

  I am having trouble with my gels....so until recently we used EtBr when casting a gel. We make a large (200 well split over four rows) 1% gel with 1xTAE. When using the EtBr the ladder came out crystal clear. We have recently switched to SybrGreen1 Gel stain and now the ladder is just a smeared mess. I read that we could used the same volume of SybrGreen as EtBr which I did at first but it was a mess. I have sincce tried various smaller volumes with a very minor improvement. Others have this trouble too and I have done a gel with Etbr since to check and it was fine so I don't think it is the gel. The ladder was originally an older one but we have also had the same problem with a new one. The bands of DNA all come out fine so I am wondering if the Sybr Green is a lot more sensitive and the ladder wells are just over loaded? I tried reducing the amount of ladder per well which also hasn't really made a difference. Should I be diluting the ladder or something?

 

ANy thoughts?

Hayley



#2 Kate Warner

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Posted 13 November 2013 - 05:29 AM

Hi Haley

 

I had the same problem when we switched from EtBr to Gel red and found I had to dilute my ladder to get good seperation. I used to use 4ul of ladder with 1ul of loading buffer with EtBr but now I use 0.5-1ul of ladder (depending on % gel), 1ul of loading buffer and then add TAE (whatever you use as your running buffer) to make it up to 5ul. Gelred is much more sensitive than EtBr hence the poor seperation, so you're probably having the same problem. So I'd suggest playing around with the amount of ladder you add or even switch ladders.

 

Hope it helps



#3 hjb60

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Posted 13 November 2013 - 06:52 AM

Hi Kate,

 

  Thanks for your reply! Our ladder already has loading buffer in it but what I might do is use different volumes of ladder and make it up to 5ul with TAE. That sounds logical! Thank you!



#4 Kate Warner

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Posted 13 November 2013 - 08:57 AM

You can definitely try that as long as diluting the ladder doesn't reduce the loading buffer meaning your ladder will float away (you can always add a bit more loading dye that should come with the ladder). Alternatively you can try reducing the amount of Sybergreen in your gel but this will of course affect visulisation of low amounts of DNA. 

 

We also used a ladder with preloaded dye and so I switched to one where I could add the ladder to the dye of my choice. I also had to switch to a different dye from the one that came with the ladder as I still got smearing. It took alot of messing around but I got there eventually.

 

No worries, finger crossed it'll work smile.png






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