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Conventional PCR of different dilutions

PCR Dilutions

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#1 ImNew



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Posted 08 October 2011 - 12:25 AM

Hey guys,

When I perform conventional PCR for the DNA isolated from the different cell concentration (10^9,10^8,10^7,10^6..... 10^0); only 10^9, 10^8,10^7 and 10^6 shows bands on the gel. But from the gel photo, the bands can be seen getting fainter.

My questions:

Is it possible that there are PCR products in the other dilutions but they are too low of an concentration to be seen on the gel?

If that's the case, should I increase the number of PCR cycles? (I am currently running for 30 cycles.)

Or do I have to troubleshoot my gel electrophoresis?

The conditions for my gel electrophoresis:

2% gel, 90 volts for 60 minutes.

Thank you.



#2 tea-test



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Posted 09 October 2011 - 11:24 PM

yes, its likely that are are also products in your samples with the low cell numbers. you just don't see them on the gel because they are too little.

And yes, you can increase the cycle number to 40 to get higher band intensities.

If you want to compare your initial amount of template in the samples I would encourage you to use real-time PCR, especially if you want to publish the results. It's also easier and faster because you don't need gels and you have high sample through-put (mostly 96well plates).
tea-test: The artist formerly known as Ned Land

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