How to calculate concentration of pure PCR product from primer dimer? Suppose PCR product concentration comes around 126ng/ul and the gel picture shows also the primer dimer, then how can I calculate pure PCR Product concentration(which will not include concentration of primer dimer)
I request you to please help me with this.
Thanking you !
I'll suggest you to check back with your primer and re-run PCR again. Maybe you will need to design a new primer which has less likelihood to form dimer. Usually I will only measure the PCR product concentrations if I could get single band on the gel. If multiple bands are obtained, it will be difficult and unreliable to estimate the PCR product concentration, especially when primer dimer is also present.
I'll suggest you to check back with your primer and re-run PCR again. Maybe you will need to design a new primer which has less likelihood to form dimer. Usually I will only measure the PCR product concentrations if I could get single band on the gel. If multiple bands are obtained, it will be difficult and unreliable to estimate the PCR product concentration, especially when primer dimer is also present.
What is the application for which you need to know the concentration?
Primer dimer is undesirable and often arise from poor primer design. It is preferred if you could get a better primer set, re-run your PCR to get a pure PCR product before you finally measure the concentration of your product with accuracy.
You can opt to either re-run your pcr to get single band or purify your target pcr product first. Then only you measure the concentration so that you can get accurate measure on your pcr product without interference from primer dimers.
You may not have to redesign your primer, 1st try to optimize your pcr reaction conditions such as annealing temperature, duration of extension and no of cycles, this might help you. All the best
If you will not be able to optimize you PCR you can alternatively clean your reaction product and get rid of primer dimers in this way. You can e.g. cut the desired band from the gel or use some of the commercially availabl kits like Ampure beads. Of course with this attempt you will lose some of your product but it can be useful if you do not have much options with PCR optimization. Good luck!
Purify your PCR products to get rid of the primer-dimers and then measure. You may lose some PCR product, but you will get enough back to measure it accurately. No need to cut the band out of the gel, just run the product through a column purification. You will lose less that way.
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