I'll be waiting for your results

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hey
I was just wondering if you could explain how this collection of biological matter happened on your tips

I too appear to be part of this miserable PCR contamination club -- I've been stuck for the past 3 months with contamination (I'm working with Herring Gull DNA - and I'm trying to amplify microsatellites) - and I'm the only one in my lab trying to finish my undergrad thesis!! ack!

anway, your posting caught my attention because I've noticed sometimes some collection of brown goop (sorry for the lack of scientific description there) on the end of the tips (i.e. the tip of the tips) -- I always don't use these tips if I see it, but I'd bet that there is the same goop on other tips at levels small enough that I can't see it with the naked eye

However, I've also used filter tips and I still got contamination
(moreover, I think I got nucleases when the filter tips weren't autoclaved - even though they said "sterile- so I autoclaved them, and hence you can see where contamination might have appeared on the filter tips too)

I've changed all the reagents (including the water - which is distilled H20 autoclaved in 1.5 mL Eppendorf tubes)-- one at a time and nothing has changed
I've even had all the pipettemen cleaned and calibrated by VWR PipettePro
I change my gloves or wipe my gloved fingers with ethanol in b/n handling template samples and making the mix
I've ordered completely new primers and I just diluted them and my most recent pcr was still showing dirty negatives!!

At this point, I'm pretty sure my thesis supervisor thinks I'm an idiot
but I don't think it's technique -- I've done PCR in other labs (granted it was with yeast) but I never had chronic contamination issues -- i.e. I know how to pipette, I don't stupidly place my tip in template dna and then go back to make up the mix

like others I've read about here, I'm getting to the point of desparation and sheer frustration
if you (janbrisbane) or anyone reading this, or even someone who might have worked with bird DNA and microsats, have any special suggestions I cannot tell you how happy I would be to hear them!

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hello,

Sometimes, bands occur in negative control is not due to contamination. When your primer contain primer-dimer, it will also create some bands in your negative control. The reverse primer and forward primer will bind among themselves during PCR amplification. Therefore, it is important to check the properties of primers before proceed to PCR process.
Plus, it is better to use Nuclease-Free water compared to sterile distilled water in PCR work since it will minimize the risk of contamination.
Enjoy your PCR work!!!

Thank you.

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try using UV-treated water...

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Following on from the last posting, try using someone else's water, buffer, tips etc. In a different lab.

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Hi all !

thank you very much for your suggestions -- my supervisor is still investigating new water options, but a little while ago we had a huge lab clean-up: all the bench surfaces, PCR machines, pipettemen, and tube racks, etc.... were scrubbed/soaked and cleaned with bleach to get rid of any DNA
-- I replaced all the PCR solutions again and performed the next one on a different bench in the lab and it actually came out clean!!

hopefully this lack of contamination will last!

on that note -- do you think it would be better to continuously replace bench paper all over the lab (as my supervisor thinks is the best option) or to leave the benchs bare and regularly wipe them down with a bleach solution? just wondering what most other labs do?

thanks again!

p.s. regarding the primer dimers comment -- I know that was not the case because the bands I was getting corresponded to sample bands - but thanks anyway ;o)

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we used to keep clean our benchtop by using spray of 70%ethanol, and wipe it with lab tissue paper

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yeah -that's defintely what I get the sense most labs do around the life sciences building too
thanks again!

(as a point of interest to anyone who can afford a more pricey option - for about $35 (Cdn) you can get this spray cleaner called 'DNA away' from VWR - I've never used it, but I thought the idea was cool)
(though I still think ethanol and bleach solutions would be just as effective and much more budget-appealing!)

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You can not dissolve DNA in 70% EtOH, so you will not get rid of DNA by cleaning a bench with it. (think about it: when doing precipitation you "wash" your DNA with 70%EtOH and it does not get into the solution, when you do spin column clean up mostly the last step before elution is washing with 70% EtOH and some salt solutions).

70% EtOH spraying of benches can be usefull to get rid of bacteria or so, and maybe other contaminants though.

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i'm always doing my PCRs with filter tips and not with autoclaved ones. sometimes i've noticed this before metioned "brown goop" on the end of our autoclaved tips too. seems to be a more widespread phenomenon. however, i never use normal tips for PCR anyway but rather for inferior operations

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