issue with taqman MGB probes
Posted 08 April 2010 - 01:08 PM
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....
Posted 12 April 2010 - 06:55 AM
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Posted 19 April 2010 - 08:43 AM
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.