Enrich the adaptor-modified DNA Fragments by PCR

[barbarella]

Hi everybody,
when you perform the enrichment of adaptor-modified DNA fragments by PCR the Illumina' protocol doesn't provide a blank control. We inserted a blank control in the PCR and we obtained a contamination. What do you think about?
Thank you!

[ieuanclay]

Was it a totally independent blank control? i.e. a water control for the PCR?
You may simply be looking at primer dimers - what size was your 'contamination'?

A good idea (at least for peace of mind), is to include a water control in the ligation reaction and take a blank gel control (a slice of similar size from a comparitive region of the gel) during gel purification, on top of the water control for the PCR, these will provide you with a good idea of whether or not your samples are contaminated or if your purifications are poor.

[barbarella]

Negative control

Thank you for your answer. We saw the size of negative control by Agilent Bioanalyzer and it was the same of the library.
To perform a control ligation is a good idea.
Thanks for your availability.

[ieuanclay]

I suspect you have contaminated samples then. The primer dimers, when you get them, are very clearly that... It is important to avoid any kind of contamination - unclean razor blades, cross contamination between gel lanes, non 'molecular biology' grade water, unclean gel tanks (UV irradiating after cleaning them is a big help), general lab cleanliness, for example setting up your library amplification PCR in the same area you made the library instead of in a dedicated 'clean' PCR area.

Hope it goes better next time,

Ieuan