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PCR triplicates versus one reaction

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17 replies to this topic

#16 molbio1234



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Posted 27 March 2011 - 05:08 PM

I am getting the feeling my question in the above post is more of a trial and error, which is a pain :blink:. Does anyone have any guidance or suggestions ?

#17 Adrian K

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Posted 28 March 2011 - 09:33 PM

No reference this time.

Or if your research was really tricky and you had to put in x number of copies of your DNA, then how to go about figuring out what x is to amplify your target correctly and avoid PCR bias.

If you know your gene sequence, design a primer. If you can extract and purified your DNA, make it in 10pg concentration. Do a 10x dilutions till 1fg level.
Run your PCR in gradient.

You have to do all the above (unless I missed out something...) in order to answer your question. You will know the minimum template DNA required for that particular PCR to work. No general or common or fixed number of amount.

I not sure what you mean by PCR bias. Bad primer design: unspecific amplification.

the effect of a low number of copies of DNA to put into your PCR.

too low maybe no amplification
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#18 Maddie



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Posted 31 March 2011 - 07:36 AM

you could also do one amp in 100ul (basically, you dilute your DNA without decreasing the amount of starting template).

i don't understand how you can dilute the dna without decreasing the starting template amount. can you give some examples.

If you put 50ng in 50ul, you have a concentration of 1ng/ul. If you put 50ng in 100ul, you have a concentration of 0.5ng/ul but you still have 50ng in your tube.

As for the low copy number PCR, we usually increase the number of cycles in order to detect PCR product on a gel.
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