Hi, I ahve tried dna extraction and when I load DNA in agarose, I have seen dimer like dimerprimer in PCR, can anyone tell me why I saw dimer in dna extraced?
Paul Rutland
If you are just running dna with no amplification then very small blurred fluorescent smear is probably degraded rna (or very badly degraded dna). Degraded rna will not stop normal pcr but will make dna quantification more difficult
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Asif Shahzad
The presence of primer dimers in DNA-extracted samples can occur due to various reasons.
- During the PCR amplification process, primers are typically used in excess to ensure efficient amplification of the target DNA. If PCR primers are not completely removed during DNA extraction, they can remain in the extracted DNA sample and lead to the formation of primer dimers during subsequent PCR reactions.
- DNA extraction protocols often involve multiple steps to isolate DNA from other cellular components such as proteins, lipids, and RNA. If the DNA purification process is incomplete or if there are contaminants present in the final DNA sample, they can interfere with PCR amplification and contribute to the formation of primer dimers.
- Degradation of DNA during the extraction process or due to improper storage conditions can result in shorter DNA fragments that may form primer dimers during PCR amplification.
- PCR conditions such as primer concentration, annealing temperature, and cycling parameters can influence the formation of primer dimers. Suboptimal PCR conditions may promote nonspecific primer annealing and dimer formation.
To minimize the formation of primer dimers in your DNA extracted samples, consider the following steps:
- Optimize your DNA extraction protocol to ensure efficient removal of contaminants, including residual primers.
- Use high-quality DNA purification kits and follow manufacturer's instructions carefully.
- Verify the integrity and purity of your extracted DNA using techniques such as gel electrophoresis and spectrophotometry.
- Optimize your PCR conditions, including primer concentrations, annealing temperatures, and cycling parameters, to minimize nonspecific primer annealing.
- Include appropriate controls, such as no-template controls and positive controls, to monitor for contamination and ensure specificity of amplification.
- If primer dimers are still observed, consider using alternative primer designs or purification methods to obtain cleaner DNA samples for PCR amplification.
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Humaira Imam
- Firstly, the running duration of PCR is prolonged, and you tried multiple times and have still same result its mainly due to inadequately designed primer.
- Inadequate primer design: Primer designing is a crucial prerequisite step in PCR. It shows us the length, efficiency, annealing temperature and product size for the selected primers. Poor primer design increases the chances of primer dimers in the reaction.
- You should design primer again and then check results, there would not be primer dimers and you would get DNA. Bahare Khoshkholgh
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Umali Kanyaza
This may also happen if you expose the DNA sample to UV light, during extraction .
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