3
$\begingroup$

We are having trouble with agarose gel electrophoresis. It used to work a couple of months ago but now the ladder always look smeared. We switched the components (1x TBE, 100bp ladder, different type of agarose and voltage) and equipment. We also tried 1.2%, 1%, 1.5% and 2% agarose.

I would appreciate it if someone can help us.

enter image description here

$\endgroup$
  • $\begingroup$ Was the alternate ladder new, the agarose? If all your components are old or being improperly stored or handled and thus degraded, you will get smearing. $\endgroup$ – CKM Jul 19 '17 at 14:44
3
$\begingroup$

Are you sure that your TBE is at the right dilution? And that you're using TBE to make the gel and also as the buffer for running? That sort of wavy line looks like what you might get if you'd made the gel with water instead of buffer, or run it with water instead of buffer. You could also try using TAE to make/run the gel as a sanity check.

$\endgroup$
  • $\begingroup$ I second the possibility of gels cast with water instead of TBE buffer. It is a classic mistake that many people make the first time in the lab. Another classic would be making gels from a 10x TBE stock and using 1x TBE for the run. $\endgroup$ – Jeppe Nielsen Jul 19 '17 at 21:02
2
$\begingroup$

Try going even higher with the agarose concentration, as it looks like you have decent separation of the small bands and poor separation of the large bands. Also try a fresh batch of agarose.

I would suggest trying 2-4% in 0.5% steps

If you are running 100bp or smaller fragments, you should in the range of 4% agarose.

Here is a nice example of agarose ranges and minimum difference of PCR products that can be resolved. https://www.qiagen.com/mx/resources/faq?id=1b23a0b3-32b4-4c1b-bd84-49b4b6a58311&lang=en

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.