Contamination with DNA?

[Netsua]

Hi,

I'm doing an study of mutation in the receptor CNR2 and i have problems with the negative control....i put DNA in all the wells except the last one where i put water as a negative control but when i see the agarosa gel there is a band at the same position as the samples in the water position...
I have changed buffer, water, taq, dNTPs and as the primers were the only thing that didn't change we thought that they were contaminated and ask for new ones....i have used new water to reconstitute them and did again the PCR with 2 blanks and one sample....and again all of them gives a band....

I can't understand what happens...any idea?!!!

I'm about to go mad...

pd: the pipets are not contaminated either

[proatpcr]

Hello there,
the pcr pro is here!

I have had this problem many times. It is usually either a contaminated pipette (yes do not use the same pipette for plasmid isolation! either that or use filter pipette tips) or a contamination elsewhere such as a buffer or water.

You can get aerosols when sucking up plasmid etc (template DNA). Then when you do a pcr, you can get this tiny aerosol injected into your pcr!

As you could be either using a contaminated pipette to do the PCR (more likely) you could also rarely get it after during loading the gel using a contaminated pipette then (ethidium is pretty sensitive).

Please let us know if it was the pipette or something else.

there is also a funny video here on pcr contamination check it out

[Netsua]

I get it!!!!

In the end it was the primers...... i did again the pcr: one with the old Fwrd biotilinated and the new Rwrd primer and other blanks with the bio and the Rwrd new primers...and the first ones were contaminated but the news not.....

I'm happy cause i can finish the study but i still can understand what has happened cause i had already done the pcr with the new primers and they seamed to be contaminated.... maybe i used the old ones thinking to be the news one but i was absolutly sure that i used the correct ones....but is the only reasonable explanation that can be given....well it's this or the bad karma

pd: very funny the contamination video