Mycoplasma detection with ATCC kit - (Oct/04/2013 )

Hi folks,

I am using the ATCC mycoplasma detection kit 30-1012K and am getting a band that is higher than their included positive control.

Does anyone know if that is indeed a positive or some non-specific amplificaiton? I don't get this 450bp band everywhere, just in some cells, but the positive control runs about 350bp, which is confusing the heck out of me.

Some cells, presumably the infected ones, do give the same size band as the positive control as expected.

Does anyone else get this higher 450bp band, and does anyone know if it is really mycoplasma contamination? I wanted to send it for sequencing, but can't since the primers are proprietary.

Thanks for your help

Vikky

your ladder looks strange from middle to top. I would run the gel one more time if I were you. Most kits I have seen are based on multiplex PCR, this one must be the same since it uses universal primers from different strains. I'm guessing if it's based on multiplex then it might give you band at different length? although most of the primers are designed for conserved regions on the genome or 16S rRNA. The other questions is that, did you run PCR with the media alone? I didn't check the protocol.

You can also check mycoplasma contamination with DAPI, it just needs a little experience to see the blue contamination.