contamination issue in -RT control - (Aug/15/2008 )

HiYa fellow PCRers,

After making cDNA (from mouse tissue), we always screen our + and - RT samples with our housekeeping primer (actin) to ensure no nonspecific product generation in the -RT. In the current batch of lung tissue, we are seeing our product of interest at the correct size (80 bp) but in the water and -RT samples, we are seeing a new band at 50 bp. If we run the gel longer, we do not see the 50bp band in the +RT samples.

Now if it was contaminating DNA, why would there be a product in the water lane and if it was a general contaminant, would we not see both bands in the +RT samples?

Any suggestions would be welcome.

AussieUSA.

PS: We are currently repeating the PCR using some +/- RT samples that have not shown this contaminant before to check the primers etc.

how is the melt curve ?? ( are u using Sybr green??) This could be Primer dimers.