6 replies to this topic

[firefly2280]

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

[mdfenko]

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?

[phage434]

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

[Ikar]

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

[Ikar]

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

[firefly2280]

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

[hobglobin]

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)