DNA degradation causes smeared bands. The curved bands tend to be caused by salts in the PCR mix and localized softening of the gel due to heating from the current applied which is also influenced by the percentage of agarose in the gel.

cDNA can be of different size whether RT stopped at different positions or that you started with a complex mixture of RNA (cellulare RNA?) so I would not bother about the smear ...

These bands look pretty decent. If you're not planning to publish the gel image I wouldn't worry about it too much. You could try making a higher % agarose, using fresh running buffer, and a lower voltage. Also, clean the comb in case there is some dried gel residue on it.

overloading and salts may be the cause of "smiling bands": try to load lower amounts of nucleic acid.

About smear, it can occur during PCR when you use too much DNA template, or when the condition for PCR are not optimal.

Alternatively, it may be degradation, but I do not think this is your case.

I would try first to repeat PCR with 1/5 of the amount you used here.

Faint and smeared bands in agarose gel electrophoresis can result from various factors. In Q5 PCR, template degradation, incomplete denaturation, suboptimal primer concentration, or excessive cycles can cause artifacts. cDNA template degradation may contribute to smeared bands, but other factors must be considered. Optimization is crucial for accurate results.

Robert Adolf Brinzer

Thank you for the reply. This is actually 2.5% gel. I may try 2% gel next time.

Didier Poncet

Thanks for the reply. RNA was extracted from a HCC cell line then reverse transcription into DNA. this procedure should be fine.

Amy Klocko

Thank you!

I used 110 volt and 2.5% gel for gel electrophoresis.

But I feel like it is hard to dissolve agarose properly with a high percentage even using microwave.

Paola Campomenosi

Thanks for the suggestion!

I will repeat using lower amounts of pcr product and larger well for agarose gel casting.

Oghenevowhero Beatrice Ereziwosa

Thanks for the answer!

I may try different polymerases such as vazyme phanta or kapa and touchdown pcr to optimize the pcr reaction.

if the agarose dissolves poorly you’ll get smeared bands. You can get low melt temp agarose. but, if you run the gel hot it will melt during running. Try mixing standard gel with low melt temp gel (half each)

Amy Klocko

Thank you.

The tank is hot for sure. I may put an ice block on top haha.