Problem with running SDS page gel - (Feb/20/2009 )
I'm running a gel right now and I'm very confused as to whether it's alright (I guess I'll find out later)
When it got past the stacking gel, there was a 'smile effect' but it was as if the dye front had split, and there was a faint line of protein and dye below the smiley dye front. Now the dye front is catching up to the faint line of protein and dye, and I was wondering what caused this problem and if it will affect my results? The bands have also spread out sideways.
Thanks for any help in advance
wait to see the result.
what are your samples in before adding loading buffer? if you are not running a gradient gel then some additives can cause band spreading during the run (detergents, lipids, salts, etc) as well as the split that you see at the beginning.
with gradient gels you often see some band spreading (path of least resistance and all that).
i do get this problem myself sometimes. I'm not really sure what causes it, but if i lower my voltage to a really low value (e.g. 30V) and run at that voltage, the smile straightens out. Not fully but more decent looking.
do you use constant current or constant voltage. I have noticed that when i use constant current, i rarely get the smile, but thats just a guess. you could try it out though.
MaggieRoara on Mar 9 2009, 01:01 AM said:
do you use constant current or constant voltage. I have noticed that when i use constant current, i rarely get the smile, but thats just a guess. you could try it out though.
your problem was caused by heating of the gel. by lowering the voltage (and current, and wattage) you decreased the heating.
heating causes smiling because a gel will get hotter in the middle than at the sides.
The smile effect is caused by areas in the gel running hotter than the rest of the gel. Just lower the voltage and it will straigten out (which you already said). But, the best way to avoid smiling is always having super COLD running buffer. Run the gel with a stir bar in the main tank to keep the buffer equally distributed throughout the tank thereby keeping temperatures the same throughout the tank as well. Any changes in the running buffer temperature will change the way the gel runs.
ALSO: Make sure if you are using any SDS, that it is checked for pH. Old SDS buffer will go acidic and needs to be checked for this. It will wreck the gel and the samples. What I started doing, was placing the gel tank (empty) in a rubbermade container. Place water in the rubbermade container and let it freeze. That way the running buffer will stay nice and cold during the run once it is added to the main tank. Make sense? Gels suck sometimes. I have been called the "gel queen" and I STILL have bad gel days.
sciencedork on Mar 9 2009, 01:13 PM said:
ALSO: Make sure if you are using any SDS, that it is checked for pH. Old SDS buffer will go acidic and needs to be checked for this. It will wreck the gel and the samples. What I started doing, was placing the gel tank (empty) in a rubbermade container. Place water in the rubbermade container and let it freeze. That way the running buffer will stay nice and cold during the run once it is added to the main tank. Make sense? Gels suck sometimes. I have been called the "gel queen" and I STILL have bad gel days.
hey thanks, i tried out using cold running buffer and lowering my gel run voltage. I also put my gel tank in a ice box with ice and one of those gel packs you can freeze to -20, u know the kind that you get when Abs are delivered.
Gel looks very pretty...
I put my sds in a 37 degrees bath before using. it dissolves all the excess ppts.
I always use 0.02A/mini gel during electrophoresis and I dont have smily bands