smears on gel.. - REASONS (Sep/06/2006 )
just want to know, at what cases a smear may appear on the gel...
is shearing of DNA is one case..gel composition?
Using vodka jelly instead of agarose?
Just a guess
Causes that i know off,
Bad DNA. Sample is really a smear (composition of many fragment sizes - due to bad PCR or DNA degredation). Or DNA is too heavily contaminated with proteins/BSA. Or DNA is suspended in solution with too high salt concentration.
Bad gel - bacteria have made a home in the agarose and has degraded DNA while it was running. Also if the gel is made up with incorrect buffer concentrations, the DNA can be smeared.
Bad loading buffer - bacteria have made a home in loading buffer and thus degrading DNA during run.(a problem of glycerol and ficoll based loading buffer.
Bad run conditions.
DNA was run too fast too (Too high voltage/amp) or in the case of very small bands (<100bps), run to slowly (DNA has time to move by diffusion) or gel not dense enough to slow down these tiny bands.
Running buffer was made up worng (usually too concentrated)
Lane over loaded, too much DNA was run on the lane.
And lastly though not real reasons; Insufficient run time. If the DNA has not run far enough to be saparated. And if DNA sample has lots of RNA contaminants, then degraded RNA will look like a smear.
Lastly a dirty eletrophoresis box, which has stuff growing in it all weekend.
I can't think off anything more right now. But hope this helps.
thanx alot...that helps
do gas bubbles have something to do with smearing?
No, not really.
If the DNA has to pass near or through a bubble, streaking and malformed bands will occur. However this too is influence by the size of the bubble and the type of gel box in use. Vertical gels are more resistant to problems caused by bubbles, mainly because the bubbles form at the very surface of the gel, Horizontal gel on the other hand, because of the apparatus setup, the trapped bubbles actually from cavities within the gel slab. Holes in the gel isn’t a good thing.
P.S: Malformed bands are also cause by running DNA suspended in too high salt concentration solution... the more salt the DNA has, the more malformed the band becomes, until it starts to look like a smear (with a tail pointing upwards). Degraded DNA have tails that point downwards (because it is broken into small fragments)