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Problem with Real-Time Pcr

Problem with Real-Time Pcr - PCR - Polymerase Chain Reaction Forum

Problem with Real-Time Pcr - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 06-02-2010, 05:28 PM
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Default Problem with Real-Time Pcr



Hey there!!
I've got a problem and I'd like to hear your opinions..
About a week ago I start doing, real time pcr...
Because it's my first time with real-time, and because I'm a student, I'm not familiar with the method yet...
The problem is that almost every time, I have contamination in water(no template control)! Why does this happen,even if I'm pretty sure that I don't do something so wrong(such as, passing with the pipette over the strip etc) that might cause the contamination??
PS: I use SYBR Green(which works fine) and not probes....
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Old 06-02-2010, 07:57 PM
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Default Re: Problem with Real-Time Pcr

Here in the lab some dudes had the same problem, and when I asked 'em what happened, they told me they had problems with the primers and gave me this tips:


- Check if the primers overlap.

- Do you resuspend liofilized primers and the same day you run the PCR? Sometimes it's recomendable to wait from a day to another to let all the DNA aggregates in the liofilized to dissolve completely.

- Check if the water is contaminated. Even the smallest amount of DNA is detectable with real time PCR. You can expose the water you want to use to the UV light to make any contaminant DNA crosslink. This way it cannot be used as template in the reaction.

Hope it helps ^^

Last edited by SebaQ; 06-02-2010 at 07:59 PM.
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Old 06-02-2010, 08:37 PM
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Default Re: Problem with Real-Time Pcr

SebaQ, first of all thanks for your answer and your advise!!
The weird is that,at the same time,another person was doing the same real time(in order to compare our results) and she was using exactly the same primers,dNTPs,SYBR Green,ROX etc....and of course she didn't have any contamination! So I'm wondering that...either I'm so unfortunate that I used the contaminated water or I don't know a thing about real time

- The primers are kept in the refridgerator and they are ready to use.The only thing we do,is vortex then and sort spin them...
-I'm gonna expose the dH2O to the UV and try again to see if anything will change...

(I apologize if my English aren't well,but I'm trying to do my best!)
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Old 06-02-2010, 10:12 PM
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Default Re: Problem with Real-Time Pcr

Relax about your english... mine ain't the big deal anyway xD

Well, if your companion did exactly the same and had no contamination, maybe you could check the reactives you didn't share, like water, pipette tips, etc.

Here at the lab, in order to avoid contamination and inespecificities, people prepare the real time PCR mix inside a laminar air flow, with sterile pipette tips, UV-exposed water and with most precautions.

Hope you get good results ^^!
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Old 06-03-2010, 05:52 PM
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Default Re: Problem with Real-Time Pcr

Thanks again!!
I'll do tommorow whatever you told me....and I hope that everything will be okay!
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Old 06-04-2010, 04:57 PM
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Default Re: Problem with Real-Time Pcr

Problem solved!!
Finally there wasn't any contamination...
SYBR Green is non-specific and probably detected something else.
They told me that, if there was contamination,the Tm should be the same as the Tm of my samples....
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Old 06-04-2010, 07:07 PM
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Default Re: Problem with Real-Time Pcr

Quote:
Originally Posted by SnWhite View Post
Finally there wasn't any contamination...
SYBR Green is non-specific and probably detected something else.
That "something else" is what it's called contamination. If there is no DNA besides the one you are putting into the reaction, what could SYBR Green get attached to?

Quote:
Originally Posted by SnWhite View Post
They told me that, if there was contamination,the Tm should be the same as the Tm of my samples....
That could do for cross contamination between your water and your template DNA, but you can't say anything about unknown DNA that could have been somewhere else, because you don't know the sequence, and therefore you can't predict what could happen with your primers and that DNA in a PCR round.

Analyzing both your product Tm and the contamination's Tm can give you the answer, because if it has a different Tm, it means you amplified something from a different sequence that came from a DNA you aren't working with.

Best regards ^^!

Last edited by SebaQ; 06-04-2010 at 07:12 PM.
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