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primer dimer


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#1 cece23

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Posted 26 August 2009 - 06:53 AM

Hi

I did a Real time using three cDNA dilution (1, 10^-1, 10^-2) in order to test the quality and efficiency of a couple of primer. cDNA was obtained by a RT of the total mRNA (so cDNA include all the transcriptoma). I checked the concentration of the dilutions with the nanodrop and there weren't mistakes. I had no problems with the first two dilutions, in fact I did a melting curve and the picks were good for both of them. Ct were respectively 21.5 and 25. The third dilution instead forms primer dimer and no (or just a little bit) specific product. I think the target gene is enough concentrated in this dilution because the second one has a Ct of 25.

Why this happen? What can I do to solve this problem?

I tried to use 2 primer concentration, 300 nM and 150 nM, but I saw no difference.

Any suggestion?

Thanks in advance

#2 k_undertoe

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Posted 29 October 2009 - 11:04 AM

Hi

I did a Real time using three cDNA dilution (1, 10^-1, 10^-2) in order to test the quality and efficiency of a couple of primer. ..

The third dilution instead forms primer dimer and no (or just a little bit) specific product. I think the target gene is enough concentrated in this dilution because the second one has a Ct of 25.


It sounds like your primers are just not efficient at that lowest dilution, which is the point of running standard/dilution series, to check at what template concentrations your primer works/gives linear amplification. This happens all the time. Primers are in theory easy to make on paper, but harder to get to actually work in real life.

If you really think your primers are great, I would first run a smaller dilution series, with dilutions factor of 10 (so 1, 1/10, 1/20, 1/30, 1/40 etc up to 100 where your reaction failed) to see if it is your primer being less efficient.

You can further optimize your reaction conditions by a number of ways, change the annealing temp to go lower and maybe get the product at that third dilution, but maybe you will also get random product/less specific product (get more random priming). do a 'temperature gradient' for the Tm around your primers. There are many sites that go into detail about how to optimize, but that is what I'd look at first.

#3 anfernee

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Posted 17 November 2009 - 04:36 AM

Do you have the false peak in your NTC? Check the melting curve and if you have it in the NTC you probably need to redesign the primer. Or perhaps my stupid idea-in my experiment I have primer dimer too.But because i do not have much choice to redesign the primer I add one step to collect the signal at 75 oC after 72 oC extension.I do not know it is right or wrong cos I am still waiting for the reply for this!




Hi

I did a Real time using three cDNA dilution (1, 10^-1, 10^-2) in order to test the quality and efficiency of a couple of primer. cDNA was obtained by a RT of the total mRNA (so cDNA include all the transcriptoma). I checked the concentration of the dilutions with the nanodrop and there weren't mistakes. I had no problems with the first two dilutions, in fact I did a melting curve and the picks were good for both of them. Ct were respectively 21.5 and 25. The third dilution instead forms primer dimer and no (or just a little bit) specific product. I think the target gene is enough concentrated in this dilution because the second one has a Ct of 25.

Why this happen? What can I do to solve this problem?

I tried to use 2 primer concentration, 300 nM and 150 nM, but I saw no difference.

Any suggestion?

Thanks in advance






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