I did PCR of 6 amplicons and when I did agarose gel electrophoresis, they all appear consistently bigger than they should be (e.g. the amplicon should be 4000 bp, but is around band of 5000 bp marker).
Sizes of my amplicons, from left to right are:
top row - 2610, 4052, 4000
bottom row - 4860, 4565, 3235
The pictures are the same, just different exposition. There are 5 identical samples differing at annealing temperature, split by marker.
The marker looks quite bad. I don't know why, I used it previously. The conditions of electrophoresis are: 1% agarose with TAE; 50 V; 90 mins
That sort of smear-y shape can be due to a few things:
loaded too much sample
dirty gel combs
removing combs too early
So, I bet if you washed & rinsed the gel casting equipment & gel rig; used fresh electrophoresis buffer; and made sure you let the agarose completely harden before removing the combs your bands will look much better.
I don't think the sizes are necessarily different from what you have predicted, pretty sure it's just the smear/wobbly bands that's making it hard to estimate the size.
Good luck!
In addition to the above if the pcr samples are in a high salt buffer then they may run slower so look larger than the quicker moving size standard so diluting the samples should get rid of some of the smearing and also lessen the difference between the ladder and the samples
Thank you for your answers.
Katie A S Burnette is it too much sample as volume or DNA amount?
It is true that I used yesterday other system than I use usually, so it could be dirty. I may have not waited long enough for the hardening.
However, it is mostly present in the samles with most of DNA, so I guess it will be the amount of DNA. Although, paradoxically, I used the same volume in larger combs than usually.
Paul Rutland I ran the same PCR as usually, so I suppose that should not be the problem.
It's too much sample as in too many molecules. Try loading a smaller volume or dilute 1:10 and load the same volume.
It sometimes help to add your DNA stain (like Ethidium bromide or Gel Red) only after the electrophoresis run is finished.
(I.e. not to add the stain yet while preparing the gel or with the sample, but immerse the finished electrophoresis gel into the buffer with Ethidium bromide or Gel Red).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA