contamination vs primers-dimers

[Anabel]

How can I differenciate DNA contamination versus primer-dimers formation working with a Taqman probe? Although I cleaned up everything and I changed reactives, I am having signal in the negative control around crossing point 30.
Any suggestion?
Muchas gracias

[moleculardude]

Hey there and welcome to the forum...

DNA contamination is usually present as higher mw bands on the gel... however try using the same primers you have with new water and see if you still get a band...

I would be careful of the pipettes you are using as they could easily have small droplets form mini preps or other forms of DNA (bacteria)...

try using barrier tips (filter tips) and see if the problem goes away...

it is difficult to assess however if you are getting primer dimers they may be present also in your + control lane... please inform us more about your situation or post a gel image here...

thanks

[danfive]

Primer dimer will go away if you reduce the primer concentration, or optimize for MgCl2. Try running the pcr reaction on agarose gels and determine if the contaminant is primer-dimer or dna by looking at the size.

[krichar214]

You could also try treating your plasticware and solutions with short wave UV light for about 5-10 minuted before adding the enzyme. Don't leave your stuff under UV light for long periods as something leaches out of the tubes and kills polymerases after leaving Epp tubes and water in platic containers in hoods with UV light for several days. This does not seem to be a problem with the aerosol barrier tips - I leave them in the hood under UV light for weeks!

[kiki06]

Hello everyone,

very good information on real time primer dimers here thanks for posting.