lousy agarose gel - (Feb/27/2006 )

preparing something as simple as an agarose gel (0.7%) has become an adventure by itself . all my plasmid preparations (alkaline lysis and kit isolation) dont show good clear bands though i make no mistake in the preparation(i am following a standardized protocol).either they appear too intense or too faint. also the other forms of plasmid (linear,nicked) appear as a smear. it is very frustrating too see an entire days work gone waste because of a lousy gel preparation. could anyone please tell me the best way to prepare a gel?

what should i look for when preparing a agarose gel?
how long should agarose be melted?
how to recast a gel?

thanks

you are using buffer (1X) and not water, right?

um, agarose must be completely melted. completely. you can re-cast by pulling it out of the frame, remelting, and repouring. did I mention it had to be completely melted? You can accomplish this with either a hotplate (while stirring gives good results, it seems to melt a bit more evenly) or a microwave (think low power setting; boil-over sucks and watch your ethidium bromide contamination inside)

it sounds like possibly you are seeing some degradation in some of your samples, and that will not be because of your gel. (the smearing, the inconsistency). why do you think it is the gel's fault?

oh, hey, if you are going to re-cast, if you have loaded any DNA into the gel, you must run it off completely. and if you use this method, please be aware that your new gel may not be properly buffered (components depleted during the run). if you are not on a REALLY tight budget, it's a good idea to start over if you have put any DNA in the gel or if you have run it for a long time.

good luck. you sound very frustrated

you are using buffer (1X) and not water, right?

um, agarose must be completely melted. completely. you can re-cast by pulling it out of the frame, remelting, and repouring. did I mention it had to be completely melted? You can accomplish this with either a hotplate (while stirring gives good results, it seems to melt a bit more evenly) or a microwave (think low power setting; boil-over sucks and watch your ethidium bromide contamination inside)

it sounds like possibly you are seeing some degradation in some of your samples, and that will not be because of your gel. (the smearing, the inconsistency). why do you think it is the gel's fault?

oh, hey, if you are going to re-cast, if you have loaded any DNA into the gel, you must run it off completely. and if you use this method, please be aware that your new gel may not be properly buffered (components depleted during the run). if you are not on a REALLY tight budget, it's a good idea to start over if you have put any DNA in the gel or if you have run it for a long time.

good luck. you sound very frustrated