Hi everyone!

I'm having trouble with my Internal Control in a real-time PCR assay. The kit protocol says to add the IC "directly to the sample/lysis mixture" during DNA extraction, so I'm currently adding it right at the first step when I start tissue lysis, but when I do this with tissue samples (skin biopsies), my IC comes up after Ct 30, which fails the kit´s validation criteria.

When do you add your Internal Control during tissue extraction to get good amplification - is it okay to add it later in the process (like after proteinase K step) instead of at the initial lysis step?

The negative extraction control shows the same high Ct problem

Thanks for any advice!