agarose gel electrophoresis - problems in marker migration (Apr/30/2007 )

the markers in my gel seem to be clustered. the products resolve better when compared to the marker. i run my gel for 1 h at 50V. im using a minigel with 1% agarose for the electrophoresis in TBE buffer. . anybody help please.

Erm... I personally think 50V is way too slow. Try to run at higher voltage. 80-100V should be fine.

If the problem still persists, change the marker. =)

It's the voltage/distance that matters...
How big is the gel you're using and what ladder are you using?

sometimes marker's band clustered for rough apply of marker in the gel

Perhaps you're loading too much ladder? Titrate it down by halves, eg, try a lane of 1x, then 1/2 your normal amount, then 1/4 and 1/8. Too much DNA can make the bands blur together.

Also I would seriously try 100v, unless you like having a lunch break while you run your gel I run my gels at 250v for 10 minutes!

Perhaps you're loading too much ladder? Titrate it down by halves, eg, try a lane of 1x, then 1/2 your normal amount, then 1/4 and 1/8. Too much DNA can make the bands blur together.

Also I would seriously try 100v, unless you like having a lunch break while you run your gel I run my gels at 250v for 10 minutes!

Not with SB as the buffer:
http://www.protocol-online.org/forums/inde...showtopic=17703

300-500v is recommended but my machine can't do that

So it is a different buffer composition. I bet it is like superbly expensive isnt it?
TBE only can reach up to 140V max.
TAE cant go more than 120V.

It's half the price of Tris and you use 1/8th as much, so it's 1/16th the price, just on tris alone... there's no acetate or EDTA so you save a bit of money there too. Most people aren't using it simply because it's new and they haven't heard about it.

Thanks, Zouden. It is really a good buffer after all.
Just wondering how many times can I recycle. Haha.. I might try to talk to my supervisor about it. Perhaps he will let me use this SB buffer.