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issue with taqman MGB probes


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#1 leonine

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Posted 08 April 2010 - 01:08 PM

Hey hi,
Im new to this forum.
I had some issues with the taqman MGB probes.
For my project, I need to design 5 primer-probe sets that can be duplexed, to allow detection of a fungi called Fusarium. Since they were Taqman probe sets, I picked SYBR green primers from literature and designed probes by aligning teh region in between. this strategy worked for 2 sets of primers and probes, however recently i ordered three new ones & they refuse to work at all. I made new stocks of DNA, ran them on gels to see whether they are the problem.
Then I did SYBR green runs using the primers, and they showed Ct of >35, so I made new primer stocks (mind you, they used to work before). After making fresh stocks of everything I ran the probe reactions again & nothing showed up. Could you please please pleeeeaaseee tell me what is going on, and why am I getting such inconsistencies with every probe taqman probe run?
i can supply details if something is not clear....
thanks

#2 Trof

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Posted 12 April 2010 - 06:55 AM

SYBR primers may not be always suitable for Taqman assay. Usualy Taqman assays have shorter amplicon than SYBR. There seem to be some problem in the probe design, if the primers work with SYBR only. How did you design the probes, did you check for secondary structure and primer-probe interactions?

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#3 leonine

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Posted 19 April 2010 - 08:43 AM

Thanks a ton for replying!

Well, I had a set of published SYBR green based primers. The amplicon was short enough (about 70bp-80bp long), so I designed the probes by aligning that 70bp-80bp region (in between) and designing the probes. I used Primer Express software and it gave me the optimum probes. The GC content, temperature and everything else was fine (in theory) ;) , but when I am doing the PCR, I dont seem to be getting any results or quantification. :huh:




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