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question on pcr product in control DNA

question on pcr product in control DNA - PCR - Polymerase Chain Reaction Forum

question on pcr product in control DNA - 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 04-21-2010, 03:09 AM
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Default question on pcr product in control DNA



Can someone please help me on this

I am working on this pcr

setup where I have three pcr

primers that will amplify 3

exonic regions of the human

genomic DNA given by a customer.

The customer didn't submit any

control dna, so my boss said to

use any control DNA I have

available. I did so, using a

bacterial dna (S. aureus strain) as my control, and

I have product in the control

dna from each of the 3 primers.

what I don't understand is how

the control dna gave pcr product

when it is a different species

from my customer dna and also

because the pcr primers amplify

specific exonic regions, how did

the control dna also show amplified material?
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  #2  
Old 04-21-2010, 11:50 AM
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Default Re: question on pcr product in control DNA

What kind of control are you planning to do?
A positive control is to indicate that the PCR process is working perfectly with all the components added (excluding the primer and template), so having a PCR product is a good thing. While a negative control (minus a key ingredient) indicates that there is no contamination, which I don't think is the control you're conducting in the experiment above.

Amplification happens when the primers bind to a specific site on the DNA template of the sample. Assuming that you practise good laboratory techniques, there is no contamination/carry over and the PCR is working perfectly, it could be due to:

1. The primers are non-specific and there happens to be a similar site on the bacteria genome which where one or both of the primer pairs could anneal to.

2. The primers are targetted to an important gene which is conserved to some degree in bacteria and humans.

3. Your positive control bacteria genome has been contaminated with DNA of some other organism (not necessarily human).
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  #3  
Old 05-04-2010, 06:47 AM
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Default Re: question on pcr product in control DNA

Right on, write on about whatever you feel best.
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control , dna , pcr , product , question


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