Problem with agarose gel with BrEt

[Mrenrisco]

Hi everyone,

I'm an undergraduated student and I have been extracting DNA from seeds successfully for a couple of weeks. But when I try to visualize the quality of the DNA extracted using agarose gel with BrEt, my gel gets orange. The DNA was extracted according Doyle & Doyle protocol. I'm adding 3ul of BrEt for each 50ml of agarose (I'm adding BrEt in the gel).

This souldn't happen. Can anyone help me? I'd like to solve this problem before my professor request me to show my results

 

As a loading buffer I'm using a solution with bromophenol blue and sucrose

Edited April 3, 2019 by Mrenrisco

[hypervalent_iodine]

I was trying to think of what colours mine were under UV and realised that our imager only does black and white, and I use orange glasses and a Safe Imager when I am cutting gels. Google suggests that orange is reasonably normal though, so I’m not sure you have anything to worry about. @CharonY would know better.

[CharonY]

IIRC, depending on the precise excitation wavelength the emission of DNA-EtBr is ~600. However, if I were to say anything, I would mention that the pocket for the marker seemed to be damaged and could benefit from a longer run time. :p

[Mrenrisco]

8 hours ago, CharonY said:

IIRC, depending on the precise excitation wavelength the emission of DNA-EtBr is ~600. However, if I were to say anything, I would mention that the pocket for the marker seemed to be damaged and could benefit from a longer run time. :p 

thanks for the answers

"pocket for the marker seemed to be damaged" could you explain it more precisely?

[CharonY]

You see the marker to be a bit wavy? Usually that happens when the comb is not pulled out smoothly, creating a jagged edge (can also caused by incomplete polymerization).

[Mrenrisco]

53 minutes ago, CharonY said:

You see the marker to be a bit wavy? Usually that happens when the comb is not pulled out smoothly, creating a jagged edge (can also caused by incomplete polymerization). 

oh thanks,

I'll try to be more careful when pulling out the comb.

But this marker is old, my professor asked me to test it, that's why I let it run for a short time.

 

 

[TeslaTurbine]

Your gel being orange is totally normal. If you really wanted to, you could try using less EtBr, but its unlikely it will completely get rid of the orange tint. You are seeing the unintercalated EtBr. EtBr increases its fluorescence 25x when bound to dsDNA. If you have enough EtBr you will see it when its not intercalated too. You can also see that the bottom of the gel is not orange. This is because EtBr has a positive charge and migrates towards the negative electrode. If you were to publish an EtBr gel, you would use a software to filter out the background and turn it black and white anyway.

 

Edited April 9, 2019 by TeslaTurbine
Duplicate sentance