I am running a qPCR with 10^8, 10^4 and 10^3 CFU/mL of bacterial samples. The amplicon size is ~800bp (which cannot be reduced). The extension time is 45s (I reduced from 1min). I reduced the cycle to 35 also but when I ran those PCR products, it shows multiple bands for low concentration samples. I tried with many different primers. Is it only primer problem or we can optimize that?
It may be a primer problem but if you have run many primers then perhaps it is a secondary structure issue as well. You could try running a DMSO gradient 1% to 8% dmso to see if it will clean up the amplification
What do you see in your negative control reactions? Some primers will give a product that is larger than the normal "primer dimer" if there isn't much template DNA. You could try reducing the concentration of the primers.
Have you tried end point PCR and how is the primer pair performance. Do you obtain a single band.
The other option is redesign primer and chose a specific region. Is there any way to reduce amplicon size. Higher amplicon size for qPCR has a poor effeciency.
To me, it seems like non-specific amplification, you can try gradient PCR. Annealing temp also slightly differs depending on the concentration of primers/buffers. If you can share me the primer information and PCR conditions, I will be happy to troubleshoot it.
The other thing, your amplicon size is far bigger than standard size for qPCR. Please look for the possibility to reduce the amplicon size (100-200 bp).
Paul Rutland I guess that will increase the sensitivity but not specificity, right?
Katie A Burnette Negative control reactions are good without any visible band or amplifications. Looking at the graph, doesn't seems like primer dimer. I guess trying with lower concentration of primer might help. Thank you!
Vanita Malekar I have a single band for high concentration samples but not with lower concentration. I suppose the primer is hitting other loci also. As this is for a different assay, I cannot reduce the amplicon size. Thank you for the input.
Binod Gyawali I already did the gradient PCR to determine the Tm. Also, we are using high end of Tm to reduce non specific amplification. As I cannot reduce the amplicon size, I am not sure what might be the cause.
Milan Panth . DMSO is an organic compound and in an organic environment the primers will only anneal if they have great homology with the template so as well as linearising secondary structure ( usually caused by ionic interaction) as the percentage of dmso increases then only the very specific primers/template binding will happen so it should result in increased specificity and cleaner amplification but with some loss of efficiency at high dmso concentration....indeed when the concentration gets too high all the pcr reactions will stop but what you are looking for is a concentration of dmso where the most specific amplification still works but the misprimed amplimers will amplify
The other option left is extract DNA from the known amount of cells, but it is difficult to extract from the lower amount of samples. There needs to be a step for enrichment. For detection of pathogens from food samples at lower cell volume by PCR, there involves process of pre enrichment. Please refer to those methods.
The reason for the multiplicity of bands in this case is the incomplete specificity of primers. It can be overcome by simply raising the elongation temperature or changing the concentration of magnesium ions. The first way is easier.
you can change the primer concentration and you can increase the template concentration also. if still multiple bands gel. increase primer annealing time and extension time.
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