Ladders not resolving on agarose gel - (Mar/05/2012 )

Hi all

I am running a 1% agarose gel, using Hyperladder 1 (bioline). 100V for about 45 mins. Also tried 80V and 90V. It was working and resolving fine but now its not. It looks like a big smudge and my bands are running like a 'w'. I am not doing anything differently from when it worked before, although the TBE buffer is a new batch. Could it be that?

It doesnt really matter as I know where my bands should be, but I need a good pic!

Thanks

yes, it could be the new batch of buffer. the pH may be a little off, the concentration may be off, the water may be off.

are your samples in the same buffer as before?

Make sure you are making the gel with buffer, not water. A classic mistake, easily made.

yes, it definitely sounds like it you could accidentally have used water. however, a higher concentration (2x or 3x) wouldn't make a visible difference from my experience

yes, it definitely sounds like it you could accidentally have used water. however, a higher concentration (2x or 3x) wouldn't make a visible difference from my experience

thanks guys, definately didnt use water. made that rookie mistake 11 years ago! must be the pH of the water maybe or the buffer

without a picture I can think of several other possibilities:
- DNA of ladder is degraded
- too much buffer covers the gel (only a few millimetres are recommended)
- too much salt in the samples
- too much DNA in the samples
- Gel was used too early after casting (polymerisation needs sufficient time)