Agarose Gel Electrophoresis

[Nefarious]

Hi everyone.

After recently performing a simple DNA separation using gel electrophoresis.
0.24g of agarose powder in 30mls of TBE buffer is i beleive 0.8% which should give a resolution of around 500 bp -7 kb if i'm right. The largest fragment picked up was howerver 23000 bp. Any ideas what has happened?

p.s it was s mini electrophoretic separation done for 30mins at 120V

[Nefarious]

thanks for the help...

[kmunson779]

I'm not quite sure what your question is.

If you measured the size of a fragment on a 0.8% gel to be 23 kbp, then I wouldn't trust that number. How did you measure size -- by comparing to a size ladder?

[Nefarious]

two fragments 125 bp and 500 bp did not appear on the gel. My question is why did the 500 bp not appear at least when the gel should have a resolution of 500 - 7k.
we were giving a list of 8 fragments 23100, 9400, 6500, 4400, 2300, 200, 562 and 125 bp and only 6 turned up on the electrophoresis plate. I assumed the two smallest hadn't showed up. it's most likely to be the other way round?
OPINIONS PLEASE?

[kmunson779]

I'm assuming that you're giving us your size ladder, or that you didn't run one.

If I expected to see 8 bands and only saw 6, I would investigate these possibilities:

1) The gel ran too long and some of my bands ran off the bottom.
2) The gel ran not long enough, or doesn't have sufficient resolving power over the full range of my sample, and some of the bands are sitting on top of each other so I can't tell them apart.
3) I used ethidium bromide staining by adding it to the gel pre-run, and my lowest bands are invisible because the EtBr migrates in the other direction.
4) I stained the gel post-run and left it too long, so my smallest bands diffused out of the gel.

[fatima]

or to continue on the list of kmunson, you didn't have enough DNA .So, try to load more of the sample

[pearson]

Does anyone know how Agarose Gel Electrophoresis can be used to separate DNA fragments based on their size?