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PCR conditions with three primers


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#1 misha09

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Posted 23 February 2009 - 11:52 AM

Hi,

I am trying to standarize PCR conditions for microsatellites. I include three primers in my PCR reaction, a pigtailed primer, a tagged-primer and a fluorescent labeled primer "the tag" (CAG tag). At the first stage I work with the pigtailed and the tagged-primer each at 0.4 micromolar, I perform MgCl2 titration from 1.5 to 2.5, at this moment I obtain the PCR product of the desired size, the problem arise when I include the fluorescent labeled primer (Usually I include it at 0.4 micromolar and reduce the tagged primer to 0.04 micormolar as suggested by most of the authors), I cannot obtain the product or obtain an unspecified product with the same amplification conditions used for pigtailed-tagged primer pair. I test with the addition of BSA, Betaine, DMSO but nothing works!!!! I also try touchdown PCR protocol but the result is the same :blink:

My fluorescent labeled primers are resuspended in TLE (Tris Low EDTA), and are conserved in the dark at
-20 C.

I have 14 putative loci, at this moment I have tested 3 primer pairs and only in one I have obtain satisfactory results :(

Does anyone have experience in the optimization of PCR with tagged primers? I will appreciate any help

#2 Qundo12

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Posted 03 March 2009 - 12:19 AM

Hi,

I am trying to standarize PCR conditions for microsatellites. I include three primers in my PCR reaction, a pigtailed primer, a tagged-primer and a fluorescent labeled primer "the tag" (CAG tag). At the first stage I work with the pigtailed and the tagged-primer each at 0.4 micromolar, I perform MgCl2 titration from 1.5 to 2.5, at this moment I obtain the PCR product of the desired size, the problem arise when I include the fluorescent labeled primer (Usually I include it at 0.4 micromolar and reduce the tagged primer to 0.04 micormolar as suggested by most of the authors), I cannot obtain the product or obtain an unspecified product with the same amplification conditions used for pigtailed-tagged primer pair. I test with the addition of BSA, Betaine, DMSO but nothing works!!!! I also try touchdown PCR protocol but the result is the same :(

My fluorescent labeled primers are resuspended in TLE (Tris Low EDTA), and are conserved in the dark at
-20 C.

I have 14 putative loci, at this moment I have tested 3 primer pairs and only in one I have obtain satisfactory results :(

Does anyone have experience in the optimization of PCR with tagged primers? I will appreciate any help

I worked with the microsatellites but didn't use the tagged primer. Which organism do you study on? To my knowledge and experience, the EDTA is DNA polymerase inhibitor and if you work with the large genomic DNA, sometime using the very low DNA amount you can get the band. Is this possible to suspend the fluorescent labeled primers in water?? I think it might be better to avoid EDTA in the reaction.




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