11 replies to this topic

[chicken]

hi

i have a contamination of vector in my primers, and i am stuck, as  i am continusly getting pcr products in negitive controls,i have confirmed even after changing all other buffers and water and etc,

so can any one help me, is there any procedure to remove vector from ur primer main stock.



thanking  for any suggestions .

[Adrian K]

Purchase new primers?

[phage434]

Purchase new primers.

Then remember to aliquot them for when this happens next time.

[BioMiha]

In my experience it's not the primers. It just seems so, because if you change everything else, you still get the product in your no-template control. This happened to us last year and we were never able to get rid of the contamination even by purchasing new primers twice. I have no idea how this happens but I have heard many people speaking about it without really being able to control the problem.

[leelee]

"I have no idea how this happens"
The only way to get contamination into your PCR solutions (primer or otherwise) is if you put it there by using poor technique or contaminated equipment (equipment contaminated through poor technique, yours or someone elses).
Contamination is not magic!

chicken, the only thing you can do is purchase new primers, and as phage said, aliquot them out immediately.

[lab rat]

Someone isn't pipetting carefully. Use barrier tips to prevent carryover via the pipette barrel.

[BioMiha]

leelee, on 01 June 2011 - 07:51 PM, said:

"I have no idea how this happens"
The only way to get contamination into your PCR solutions (primer or otherwise) is if you put it there by using poor technique or contaminated equipment (equipment contaminated through poor technique, yours or someone elses).
Contamination is not magic!

chicken, the only thing you can do is purchase new primers, and as phage said, aliquot them out immediately.

Have you ever had this problem and been able to get rid of it by purchasing new primers? About a year ago I would have agreed with you, but we really tried everything (and no it is not bad technique) and still could not get rid of the problem. For example we were even able to amplify the contaminating amplicon in the no-template control in a different lab using their PCR reagents and a fresh primer aliquot.

[leelee]

How else does a contaminant get in to your reaction?

Did you use the other lab's pipettes, tips, etc. Did another person set up the reaction?

No, I've never contaminated my primers. I have contaminated my negative control within a reaction, but no I've never contaminated stock solutions.

Edited by leelee, 01 June 2011 - 09:24 PM.

[BioMiha]

leelee, on 01 June 2011 - 09:23 PM, said:

Did you use the other lab's pipettes, tips, etc. Did another person set up the reaction?

Yes and yes.

I (and other people I talked to) have found this to be a serious problem when you are amplifying the same amplicon over and over again. We have a very strict policy regarding PCR. We prepare the stock solutions in a separate laminar flow hood from where we add the template DNA (again in the hood) and have different rooms for pre-PCR, PCR and post-PCR and we are not allowed to go backwards (although I can't honestly say that everyone really abides by this). We always use barrier tips and have our pipettes calibrated once a year. In the project I was telling you about we used PCR grade water to resuspend and aliquot our primer stocks and a different lab mate (one who had never before handled this PCR product) performed this, so as to reduce potential contamination to a minimum, but we could not get rid of the contaminant. As I said, not even in a totally different lab. So, I am speaking from experience and not just using common logic when I say I have no idea how this happens, but it does. And I can't even give a suggestion to chicken how to handle the problem, because we just stopped the whole thing and moved on. And other people whom I talked to did the same.

[Trof]

BioMiha: Was your amplicon by a chance a bacterial gene? Some Taq enzymes produced in bacteria can have contamination by traces of host strains DNA.

[BioMiha]

It was a phage T7 gene. We did also try a number of different polymerases. The fact is that at first we didn't observe anything in the no template control. Only after a few months of qPCR work with the same amplicon did things start to get messy.

[tk2509]

I have similar "contamination" problems. Do you think that if I use the relative quantities of the unknown samples and find that they are 100 or more times higher than the controls, I can use my results?
Or if the Ct value difference between the unknown samples and the controls is bigger than 6-7 cycles?