I notice amplification in NTC with Ct value 18 cycles apart from sample. Ct sample 13, Ct NTC 31. Also, I have run a gel to check the product size. The size is similar to my product length. There was no amplification in NTC in previous run when the primer concentration is 0.2 uM. NTC amplification happens when the primer concentration is increased to 0.4 uM (Same primer working stock is used). This amplification only occurs in my reference gene, which is Gapdh. My other gene of interest is fine. Is this acceptable since it is 18 cycles apart from the sample?
Hi Poh, you used GAPDH as endogenous internal positive control. The main is to validate the reaction for target negative results. It is normal that you get high Ct for wells that not containing target gene, and low Ct for those containing the target gene due to the competition between the sets of primers and probes. For the same raison, generally a multiplex pcr is less sensitive than the monoplex.
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