I am new to running PCR gels. I loaded this gel and I thought it was fine, meaning I saw/felt no apparent punctures or spillovers to neighboring wells (see picture 1). When the gel started to run, the bands began to smear (see pictures 2 and 3) and ultimately, no result could be visualized in the gel (see picture 4). I used freshly prepared buffer when running this gel. Any ideas as to where it could've gone wrong? It looked to me like the dye had settled down at the bottom of the wells before I turned on the current, so this result surprised me.
The images make me think your gel was not fully solidified before you ran it or you ran it at too high of a voltage and kind of melted it. your ladder did run some so that is a little reassuring.
Basic tips for PCR gels
1. use the same buffer as running buffer to make the gel.
2. make the lowest % you need for good separation. 0.8% or 1%, only use higher ones if you need to separate bands close in size
3. when loading the wells there is some fuzziness like you saw that forms. try to load more quickly or leave an empty well in between, especially if you are going to cut and sequence bands.
Intermediate tips
TAE/TBE need to be run at lower voltages, so I like to use "FAST buffer" which is sodium borate based.
Recipe:
20X FAST SB Buffer
8g NaOH (200mM for 20X)
47g Boric Acid (760mM for 20X)
H2O to 1L
Run gel at 100-150V!
Sodium borate buffer for rapid DNA electrophoresis (lifescience.net)
Hello Angelica,
The band smear could be due to:
- Adding gene ruler wrongly to each sample (instead of dye).
- Another potential cause of the band smear is using a concentrated buffer for the gel, instead of a properly diluted one. This can affect the separation of the DNA fragments and should be avoided.
- Not given enough time for the gel to be completely shaped.
- High device voltage.
I hope these tips will help you.
The loading dye isn't smeared, it's separating out the 2 different dye molecules into separate bands.
Talk with your research mentor. See if you can get a good positive control to include in your PCR reactions. I'm seeing an agarose gel with no amplified PCR products, just the ladders & a few faint streaks where you loaded your reactions.
And ignore the "advice" from Mahdi, it's AI-generated nonsense that doesn't even answer your question.
Good luck!
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