Hello, I was running a PCR with various primer concentrations (I am aware that some of those concentrations are outrageously high). To my surprise the lowest primer concentration (200 nM) had a PCR product of different size. All the reactions were setup in a single PCR rxn mix and the primer dilutions were made fresh and added to the individual tubes. I am slightly puzzled by this result and wonder if someone can get me some clues, what might have happened. Thanks.
The smiling of the amplimer above 200nM suggests that there is too much salt ( primers/ntps/inorganic salts) in the sample and this is affecting the mobility of the dna amplimer. You could try running samples 800nm, 200 nM and an equal volume of 800nM product and 200 nM product. If the mixture sample runs like the 400nm sample in your gel picture it suggests that the amount of salt in the reaction mix is the issue. If not then possibly the less abundant pcr product at 200nM still has some polymerase attached to it in which case adding 1ul of 1%SDS to the sample may make the product run quicker but this seems a less likely possibility
Thank you for your answer, the idea of polymerase stuck to the PCR got me onto something. The previous posted gel contains Biotium GelGreen DNA stain, that is the reason the samples look so streaky (not the salt concentration). I ran the PCr rxns again and did EtBr post-staining, and to my pleasant surprise the PCR product with the lowest primer concentration is running to its expected size.
My lesson learned here, be careful with in-gel DNA stains.
Very well done Bettina Wittler . I had not realised that gel green and prestaining made such a marked difference. Good result. You might now minimise the primer dimer with a combination of primer concentration and a hot start enzyme/pcr
Reasons why you might see an unexpected PCR product size with a lower primer concentration, and some suggestions for troubleshooting the problem are:-
1- Primer design issues:
Check primer sequences and make sure they are specific to the target sequence and do not contain any unwanted secondary structures or cross-reactivity.
2-Use primer design tools to check for potential off-target amplification or primer duplex formation.
Ensure that primer melting temperatures are appropriate for the PCR conditions.
3-Optimize primer concentration:
The optimal primer concentration can vary depending on the specific target and PCR conditions.
Try testing a range of primer concentrations, such as 0.1 μM, 0.2 μM, 0.5 μM, and 1 μM, to find the best balance between specificity and sensitivity.
4-Ensure that primer amounts are accurately determined and diluted properly.
Template quality and quantity:
Ensure that the DNA template is of good quality and free of inhibitors that may affect the PCR reaction.
5- Check the concentration of the DNA template and adjust if necessary, as too much or too little template may result in non-specific amplification.
Optimize PCR conditions by:-
1-Optimize the annealing temperature to improve the specificity of the primers.
2-Adjust the extension time to ensure complete amplification of the target sequence.
3-Consider using a more sensitive detection method, such as real-time PCR, which can provide more insights into the specificity of the amplification.
Primer specificity:
1-Perform a BLAST search or other sequence alignment tool to check for potential off-target regions that the primers may amplify.
2-Design new primers or use a different set of primers that target a different region of the target sequence.
Non-specific amplification:
1-Unexpected product size may be due to non-specific amplification or primer duplex formation.
2- Try adding a step to your PCR protocol to assess the specificity of the amplification, such as melting curve analysis or gel electrophoresis.
3- If the above troubleshooting steps do not resolve the issue, you may need to further optimize your PCR conditions, consider using different primers, or check for potential interfering factors in the sample.
If you need anything, please contact me. Regards
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