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what is the order you follow in the run ???


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#1 nightingale

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Posted 24 July 2010 - 05:43 AM

in running amplification sessions, what is the order you follow ???
we have in the run :
NTC = only the PCR mix & water.
negative control = previously tested negative sample.
positive control.
patient samples.

in the run, do you add first ???
1) NTC, -ve control, patient samples, +ve control. or
2) +ve control, NTC, -ve control, patient samples. or
3) NTC, -ve control,+ve control, patient samples.

or another order ???


which you find it best, to reduce the risk of contamination ????
" The more you learn, the more you realize how little you know ... "

#2 ivanbio

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Posted 24 July 2010 - 05:47 AM

In my experience most people run the NTC and -ve control before patient samples. Yet, if you want the NTC to be truly a NTC, I would think it has to be run at the end of all samples. That way if you carry over any kind of contamination you would detected it in the last sample, the NTC.

My two cents.

Ivan
Carlsbad, CA

#3 perneseblue

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Posted 24 July 2010 - 06:47 AM

in running amplification sessions, what is the order you follow ???
we have in the run :
NTC = only the PCR mix & water.
negative control = previously tested negative sample.
positive control.
patient samples.

in the run, do you add first ???
1) NTC, -ve control, patient samples, +ve control. or
2) +ve control, NTC, -ve control, patient samples. or
3) NTC, -ve control,+ve control, patient samples.

or another order ???

which you find it best, to reduce the risk of contamination ????


I would run

NTC, negative control(1), patient samples, positive control, negative control(2)

Contamination during PCR preparation will show up in negative control (2)

The order which you run your PCR does not reduce the risk of contamination. The order does give indication that contamination has occurred. If you want to minimise contamination you will have to improve your PCR preparation methods.
May your PCR products be long, your protocols short and your boss on holiday

#4 nightingale

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Posted 25 July 2010 - 09:09 AM

thanks ivanbio & perneseblue for the replies :)

The order which you run your PCR does not reduce the risk of contamination. The order does give indication that contamination has occurred. If you want to minimise contamination you will have to improve your PCR preparation methods.


grateful ...
" The more you learn, the more you realize how little you know ... "




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