Overloading of gel

[charismafull]

How can I solve my problem, I need to digest 15 ug of DNA and the total volume I got from the entire experiment is 120ul(includes DNA to be digested, the digesting enzyme volume, buffer and water). My agarose gel would only allow me to load 60ul. What should I do?

[kmunson779]

1) Load multiple lanes
2) Precipitate your DNA and resuspend in a smaller volume

[charismafull]

I don't get it when you say load multiple lanes? This will disrupt my process coz I split my entire sample. But the second option of denature of DNA and resuspend it might be a better idea.

[charismafull]

I don't get it when you say load multiple lanes? This will disrupt my process coz I split my entire sample. But the second option of denature of DNA and resuspend it might be a better idea.

[charismafull]

i mean precipitate the DNA not denature. I'm sorry for the wrong use of words ;-)

[kmunson779]

I mean, if each lane can hold 60 uL, add loading buffer to your 120 uL and load 3 neighboring lanes of the gel with equal amounts of your digest. Then, in your next step (I assume a gel purification?) pool the three lanes as soon as it is practical.

There are problems with both my suggestions: splitting the digest will make your next step a little more complicated with the re-pooling, and will take up more reagents as you have to do the first part of whatever you're trying to do 3 times. However, precipitating your DNA and trying to run it all in one lane can cause issues with gel migration; generally I see a decrease in effective resolution between sizes of fragments when the lane is overloaded (what would be nice bands can become smears).

[jgleema]

A Mini elute column can be used just to concentrate the product.

[Wandrews963]

This may not be any help at all but could you use tape on two lanes of your agarose gel to make one big lane that woudl hten hold 120 ul? We did this in my old lab when samples were too big and things worked ok. Although there is a chance if something goes wrong you've lost all your sample which should be taken into consideration.

[luisillo]

Can you make a bigger gel and make the wells wider so your entire 120 ul fit in?