Gel electrophoresis - (Jan/10/2011 )
Dear all,
I have two question about gel electrophoresis, the first one is:
if I try to keep the gel after running to save my sample for long time, how can i keep it?
And the second question is:
If I try to cut specific DNA band from the gel, then at which voltage I should run the gel? to make long distance between bands..
Thank you
Hola,It isnīt good idea store the gels after running, because if you store it in a wet chamber to avoid dryness, the DNA band will difuse.Second if the digestion has been complete you can run any time enought to see bands focused, but itīs good run the gel as far as possible to clearly separate linear DNA of little background without digest, because few molecules of non digest DNA will give you high background after transformation. At the time of cutting bands adjust the cut as much as possible to the band edge. Buena suerte
Why don't you just extract your DNA from your gel straight away? It is a very very short process, particularly if you use a kit (and they aren't that expensive)
But if you must keep it in the gel, then I would suggest at least excising the band and then freezing until you are ready to proceed.
Keep in mind your gel will not be nuclease free....
Keeping the gel will cause the bands to diffuse after a short period, if you cannot do the extraction right away then I would agree with lweelee to at least cut the band out as soon as you can and store it in the freezer.
Voltage and time for the gel depends on what size bands you are cutting out, and the size of any other bands that will also be on the gel. the smaller the bands you are looking for the shorter time you can run for, heavy bands take much longer to run to distinguish between sizes. If your band of interest is 3.5kb but there is an irrelevant band at 3kb, then you may need to run for linger to separate them more than if the only contaminants were of a much heavier size.
I found an awesome new way to cut my gels.. Instead of hassling with a potentially non-sterile razor blade I use these disposable X-tracta tools and its 10x easier.