University of Hawaiʻi at Mānoa
Question
Asked 1 March 2023
What causes the smear below the band in electrophoresis?
When the PCR product was electrophoresed, such a band was visible and smear was seen under the band.
Cycle conditions were (1) 94.0°C for 3 min, (2) 94.0°C for 10 sec, (3) 55.0°C for 10 sec, (4) 72.0°C for 7 sec, (5) 72.0°C for 1 min, and (6) 20.0°C for 10 min.
The number of cycles is 40 cycles from (2) to (4).
The swimming time was 15 min.
Gels were 3% agarose gels.
I would like to know what causes such smears and how to improve them.
Most recent answer
A smear under the band in electrophoresis can be caused by multiple factors, including incomplete amplification, excess template DNA, too many cycles, inappropriate annealing temperature, and degradation of the DNA during the PCR reaction.
In your case, the PCR reaction has a short extension time of only 7 seconds, which could contribute to incomplete extension and result in smearing. Additionally, the number of cycles may be too high for the specific PCR reaction, leading to over-amplification and the production of nonspecific products. The annealing temperature could also be contributing to nonspecific amplification, as it is only 55.0°C, which may be too low for the specific primers being used.
To improve your results, you may consider optimizing the extension time and annealing temperature for your PCR reaction, reducing the number of cycles, and adjusting the amount of template DNA used. Additionally, you could try using a different DNA polymerase or optimizing the PCR conditions to reduce degradation of the DNA during the reaction.
These video playlists might be helpful to you:
All Answers (7)
Okayama University
Thank you for your answer.
We have tried reducing the amount of loading on the gel but it did not make much sense.
I forgot to note about the voltage, but we are using 135V.
I didn't specify, but this PCR uses the PCR product as a template and amplifies it.
And the template showed a clean band on a 135V, 3% agarose gel.
So I thought that changing the voltage would not make much sense.
Based on this, is it still advisable to change the voltage?
I'm not skilled in electrophoresis, so I'd appreciate it if you could let me know.
This smear can be some non-specific products of the size of your desired band. PCR product when used as a template does sometimes cause this problem.
Okayama University
Thank you so much for your answer.
Is this smear something that does not affect me in particular?
Also, is it possible to eliminate this smear?
well, it might affect your downstream process (if the smear is quite a bit)
You can either amplify it from the original template (if that has worked) or use it only after gel purification.
University of Hawaiʻi at Mānoa
A smear under the band in electrophoresis can be caused by multiple factors, including incomplete amplification, excess template DNA, too many cycles, inappropriate annealing temperature, and degradation of the DNA during the PCR reaction.
In your case, the PCR reaction has a short extension time of only 7 seconds, which could contribute to incomplete extension and result in smearing. Additionally, the number of cycles may be too high for the specific PCR reaction, leading to over-amplification and the production of nonspecific products. The annealing temperature could also be contributing to nonspecific amplification, as it is only 55.0°C, which may be too low for the specific primers being used.
To improve your results, you may consider optimizing the extension time and annealing temperature for your PCR reaction, reducing the number of cycles, and adjusting the amount of template DNA used. Additionally, you could try using a different DNA polymerase or optimizing the PCR conditions to reduce degradation of the DNA during the reaction.
These video playlists might be helpful to you:
