For some reason my gel today came out very weird. I ran a 1% agarose gel with EtBr added before pouring. I used 1x TAE ran for 60 using 60 V. The ladder is a 50 bp ladder and the band I am looking for is about 300 bp. This was colony PCR which I have used a lot and gotten good gels. As you can see in the picture the ladder is also blurry which make me think that it is something wrong with the gel. I have used all of the same reagents to gels with even smaller bands except the TAE is a different batch.
as your samples are a good distance from the wells it looks like the gel has been run too high a voltage and the smearing is due to heating of the gel and running too fast. Check it with a different TAE and power supply in case one of them is misbehaving. The ladder really has run a long way. Lower voltage is also worth a try and check that the current is as usual for your gels
if you want to use the 50 bp ladder i would run a higher % gel, maybe 2%, i think the other conditions (60 Min, 60 V) could be fine then for this fragment size, it really seems that the gel run too long (like above mentioned). the smearing problem could also be a result of problems in colony pcr, maybe have a look if something changed in conditions or chemicals, or in pcr program. good luck!
The fact that DNA was migrating correctly towards (+) electrode indicates that the pH of a TAE buffer was OK. There may be something wrong with its ionic strength that may be too high (which may happen, e.g. by using 10 x TAE instead of 1 x TAE). High ionic strength of an electrophoretic bufer will lead to higher currents (which could be checked) causing overheating of gel and partial denaturation of DNA. Denatured DNA migrates aberrantly in agarose. Recommendation: Make a new 1 x TAE buffer as Paul mentioned previously.
Since the band you are looking for is 300bp which requires higher concentration of agarose for its separation. Ideally 1.5% to 2% gels are used. The TAE (1X) buffer should be fine for the technique. Inorder to increase the resolution of your sample and get crisp bands, run at a lower voltage for longer time.
you should check your TAE pH before pouring on try and using casting gel, different pH also cause heat.
Dear Austin Paul Wright,
If you make delay in visualization after completing an electrophoresis, then normally the band goes so dull and blurry because as the electricity is not flowing so the staining goes diffuse through the gel.
Sometimes smear can be happen, in case of capturing a gel photo without auto-focusing technology, it always makes you band blur. Primers concentration and annealing temperature of the primers is also important to have a clear band. So, may be you can solve your problems and make something good.
Like in my gel i used a high concentrated DNA and here you can see some smears but the pcr product is seen. Here, i had used 100bp ladder and used 1.5% gel and run for 26 minutes in 150 Volt.
Good wishes for your work.
use agarose gel 2% and running buffer, freshly prepared and ladder as suggested by Dr. Islam Rabbi
I think the problem was most likely too low a voltage and too low concentration of agarose. I ran the gel again and used 150 V and 2% gel. I ran for 23 minutes and it came out beautiful.
- Badges
- Science method