Amplification in negative control during PCR

Monique

New member
Nov 16, 2015
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#1
Hello everyone!

During every amplification in my negative control I have been noticed band in NTC on agarose gel(Every day- two weeks). Negative control is same size such as positive control, and I have amplification in every sample. I can not understand what is happening, because I prepare master mix in PCR clean room, I have been changed every reagent (praimers, new lot of master mix, pcr clean water), disposable glows, filter tips, another anniling temperature...

and I m exhausted :)

I tried to minimalize enviromental factors but I don t have any idea anymore...

What are your experiences with this problem? How do you solve it?

Somebody said: routine PCR? Be honest its not exist such a thing....
 
Nov 16, 2015
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#2
Do you set up your NTC before you set up your positive control and regular reactions?



Have you also set up a NTC by itself, without setting up any other reaction?


 

Monique

New member
Nov 16, 2015
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#3
Hello!
My negative control is : 2ul nuclease free water
x ul Master mix
x ul Praimers
Everything is same such as in sample tube, just instead DNA i have water.
I set up NTC in clean PCR room. When I run gel, I have band (the same size like my positive control) in every place-including NTC.
Any ideas?
 

Ivan

Administrator
Staff member
Nov 16, 2015
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#4


Here are my thoughts:



1. Your nuclease free water is contaminated. Get a new vial

2. If your positive control is small (less than 100 bp), then maybe you are seeing primer dimers instead of an actual band

3. Do you have any samples (like negative controls) without this band? If the band is everywhere, then it is clearly contamination

4. Have you run a samples without water? It is possible that the contamination is somewhere else, like the primers or even the buffer



Good luck


 

Monique

New member
Nov 16, 2015
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#5
Thanks a lot for reply.

I changed another aliquot of water, I even used water from another trade mark but the bands in ntc are present- again the same size.

I opened a new primer set, used another primers for different assay and always have a same problem.

Sometimes I have bands in all sampels, sometimes I don't, but always have it in negative control.

It could be a primer dimer, whats the rule for it's development? Inadequate temperature?

I use primers from ISO standard method, so it has to be a good experimental design (good choice of primers set, temperature program... )

I think I will try to decrease a cycle number from 40 to 35..

Currently I'm doing a big lab cleaning with NaOCl..but maybe a crucial problem in my lab is that I don't have a separete room for PCR and electrophoresiss from place where I doing isolation of sample DNA.

I thinking about touchdown PCR, some sugestion?

I'm appreciate every idea and thanks a lot


 

Ivan

Administrator
Staff member
Nov 16, 2015
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#6


The fact that you've changed your water, and used new primers, and still see the band suggests to me the band is coming from somewhere else.



Primer dimers can develop in many different ways, including long term storage (over a year), inappropriate storage (not frozen), or simply primer design (there is too much overlap between primers so they anneal). I am not sure if you store your primers in solution together, but if you do I recommend you store your primers separate. I mean, do not mix them ahead of time. Also try not to freeze and thaw your primers too much (more than two or three times), or you will induce primer dimer formation.



Personally I do not think reducing your cycle number will do much good, but it may be worth a try. Doing a good lab clean up is a good idea. In my experience, not having a separate PCR room is not a big deal, as long as you are taking all precautions not to cross-contaminate with DNA when you are preparing your PCRs versus isolating DNA.



At this point I think it is worth trying different things, even touch down. If your problem is primer design, touch down may actually help.



Good luck




 
Jan 26, 2016
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#7

Hi,

I have the same problem with my NTC: positive results in the negative control, also I obtained bands during electrophoresis, the bp is similar with the positive control is at the same level. I changed everything, mastermix, primers, water. Also I ordered a new primer set targeting another gene and I got the same results, peaks as in case of positive control and bands after electrophoresis. Also I introduced a melt curve step which is similar sometime (but sometimes not) with the melt curve of the positive conrol. I want to know what was your problem?

Thank you in advence,

Zsuzsa