Gel extraction problem ~HELP~ - (Nov/10/2007 )
I'm having a problem whereby there is too many bands in the lane I want to extract, so much so that there isn't good enough separation if I load in a high amount of sample into the well (~20 microL) therefore I can't properly extract that band i want. However if i use a far too little amount of sample (~13 microL) there isn't enough DNA concentration for me to be able to extract the band. I've tried running the gel overnight in a 2% agarose gel at 35V in the big gel tank however I still can't seem to get the right concentration of sample and good separation. Any suggestions? Some of my lab colleagues suggested that I try to PCR the band once I've extracted it in order to increase the sample concentration. However since I am going to use the extracted band into cloning, is there any point in running a PCR? Besides, he has not tried it before and since I'm running out of time is there anyone who has encountered this problem before and has a another suggestion?
Another problem is that after extraction of some of my gels, I noticed that once I do an electrophoresis of the extracted band, it appears slightly smeared. Is that common? Or is there some technical problem on my part?
(Another quick question: What is the lowest voltage that I can run the electrophoresis at?)
Thanks in advance!
Think about the possible cutting of the fragments you do not want with another restriction enzyme -- one which does not cut your fragment.
What size are these fragments? With long (1500+) fragments DNA diffusion in gels is not much of a problem. But with shorter fragments, the separation of the bands is competing with the diffusion of the DNA in the gel, and long running times may make things worse not better.
For short fragments, resolution will be improved using Metaphor or Nusieve 3:1 agarose.
You don't need much DNA for cloning -- probably much less than you think.
More information would be helpful. What size band are you interested in and what are the sizes of the unwanted bands.
You can use PCR to amplify the segment of DNA of interest. How big is the DNA band of interest?
I like Phage's suggestion of digesting the bands with REs not in your insert. If you don't know what the unwanted bands are then use as many REs as you can. Another thing you can do is proceed with the cloning as usual and pick up the wanted clones at the screening step. Some incorrect fragments may be cloned but that's less likely because those unwanted products may be amplified by only one of the primers annealing to both ends of the fragment. The screens wil pick up the correct clones any way.