PCR contamination makes me mad !!

[valou]

Hi,

I bet this is not a very original thread but...

I started having contamination a few months ago while I was working on another project dealing with plant microsats (before that, never had any issue with PCR).
Now I am back to my own project (microbial stuff) and I still have contamination (the negative control of my PCR is positive while it only contains mastermix). The band is at 1.5kb (bacterial 16S).
In between the two projects, I cleaned the whole lab, bleached the pipettes, benches, etc...Plus, I do not use the same chemicals. The contamination seems random (ie if I prepare several negative controls some will have a band, others won't).

What else can I say ?
Please ask for more informations !

[aftabac]

sometime your primer solution get contaminated and cause problem..........

sterilize your tubes carefully and also use distiled autoclave water........

best of luck........dont loose heart..........it will be okay soon......

regards
aftab

[valou]

Hi,

everything I use is autoclaved. Primers are new, pipettes were bleached.
But contamination is still there, I just don't get it !

[butters]

i see this problem before. if you can use UNG and dutp. it will reduce and eliminate the circulating pcr product from ur previous pcr reaction. Before u go purchase the mention expensive reagent maybe u can check the UV lamp is still functioning properly or not, and use DNA away or any DNA removal solution after each preparation.

how u check ur UV lamp stil ok or not? well, just UV ur mastermix but include control (without UV) say for 15-30 mins. Then run gel and check whether the treated mastermix still able to amplify pcr. if yes maybe ur UV is not efficient

[moleculardude]

Yup,
there are several things that cause contamination even if you are clean:

Do you use the same pipettes for microbiology work, plasmid isolation from bacteria or other similar?
If so you should have dedicated pipettes for PCR. If not, try filter pipette tips when using those same pipettemans for PCR.

trust me works wonders...

Let me know what it was

[moleculardude]

Yup,
there are several things that cause contamination even if you are clean:

Do you use the same pipettes for microbiology work, plasmid isolation from bacteria or other similar?
If so you should have dedicated pipettes for PCR. If not, try filter pipette tips when using those same pipettemans for PCR.

trust me works wonders...

Let me know what it was

[valou]

Hi all !

Thanks for your replies. So, what I now do : I use a set of pipettes only for PCR, filter tips, PCR-grade water and Shrimp DNase. With all of that, things are back to normal.