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CP of real time PCR by LC480


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#1 Sonia

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Posted 02 June 2009 - 08:39 AM

My runs give high CP, always> 30 cycles. I measured the template conc. and is >1000ug/ul, why did I still have that high CP.

#2 Dr Teeth

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Posted 04 June 2009 - 06:36 AM

My runs give high CP, always> 30 cycles. I measured the template conc. and is >1000ug/ul, why did I still have that high CP.


...Not a lot of information to go on here. A CP > 30 is very high and usually seen in no template controls.
Questions then: I assume that your template was 1000 ng/ul not 1000 ug/ul! but how much template did you use? What was the 260/280 ratio? 230 ratio? Did you check your mRNA quality on a bioanalyzer or agarose gel before cDNA synthesis? What of the primers? Are you using validated primer sets or did you design them yourself? Do you know that the primers work? Do you have positive and negative controls?

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley




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