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Real Time PCR DNA Concentration

Real Time PCR DNA Concentration - PCR - Polymerase Chain Reaction Forum

Real Time PCR DNA Concentration - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 04-11-2013, 09:16 PM
Pipette Filler
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Talking Real Time PCR DNA Concentration



I have a set of mtDNA Taqman assays for various animal species and I'm trying to validate them. Because this assay is qualitative, I have not been diluting down the DNA after extraction (from muscle tissue) before PCR. The assay has a good sensitivity (~0.05% target in mixed species tissue). However, some of the assays are giving non-specific amplification at around Ct 30-35.

Could this non-specific amplification be due to running too high concentrations of non-specific DNA?

If I dilute my DNA (to 10ng per rxn for example), am I likely to lose my 0.05% LOD?

Thanks.
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Old 04-12-2013, 01:25 PM
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Default Re: Real Time PCR DNA Concentration

If it is qualitative why not trying conventional PCR (instead of real time) and running gel electrophoresis in an agarose gel? Might be an old technique but it still works.
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Old 04-12-2013, 07:23 PM
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Default Re: Real Time PCR DNA Concentration

Hi,

I need the good sensitivity. Plus, i have limited space so i want to avoid post pcr product handling and the contamination risks that goes with it.
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Old 04-15-2013, 02:43 PM
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Default Re: Real Time PCR DNA Concentration

High concentrations of Mg+ can cause non-specific amplifications. Since you are using a commercial kit I doubt you can change Mg+ concentration in the PCR mix, so I suggest you try raising the annealing temp in the thermal cycling depending how noisy is the unspecific amp. Usually raising 0.5 to 1 C in the annealing temp does the trick.

High concentrations of DNA can inhibit PCR reaction, but I do not remember having issues with specificity.

Good luck
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Old 04-15-2013, 06:29 PM
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Default Re: Real Time PCR DNA Concentration

Thats a good call - i'll try that. Thanks.
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concentration , dna , pcr , pcr assay development , pcr setup , pcr troubleshooting , real , time


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