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The iGEM DNA Kit Plate Instructions say that there is only 2-3ng of DNA per well, which is already miniprepped and in plasmids. Then it says that "there is not enough DNA in each well to perform anything but transformations".

Couldn't PCR be performed on an amount of DNA that small, given appropriate primers? Is transformation truly the only thing that could be done with that DNA? If PCR could be done on the DNA, why might they suggest otherwise?

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2 Answers 2

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You probably could do PCR on that amount of DNA, but if you screw something up you don't get a second chance. Transforming bacteria and doing a miniprep turns your 3ng into a few micrograms, which is enough to tolerate a few mistakes.

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  • $\begingroup$ A failed transformation would have the same bad result - no second chance - so that leads to the (obvious?) follow-up question: Is transformation a lot more reliable than PCR? $\endgroup$ Jan 15, 2015 at 23:18
  • $\begingroup$ Transformation with intact plasmids is usually ok. I only have trouble with transformations when ligating a plasmid together. Remember these plasmids have all been transformed into bacteria, grown, and prepped, so they are intact and ready to go back into bacteria. $\endgroup$
    – user137
    Jan 15, 2015 at 23:21
  • $\begingroup$ What's the approximate percentage of success in PCR and in transformation, in your own experience? Thanks again. $\endgroup$ Jan 15, 2015 at 23:24
  • $\begingroup$ Guessing a percentage is hard, there have been times when ligating a plasmid when I was glad to get 1 colony. If the plasmid was intact I can get a lawn of colonies if I don't remember to dilute the cells. I can't guess a percent for PCR but there are more things that can go wrong, primer design, reaction setup, purification, etc. Transformation has about 1 step, add DNA to cells, much smaller opportunity to fail. $\endgroup$
    – user137
    Jan 15, 2015 at 23:28
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Here is the feedback I got from iGEM HQ:

Yes, we estimate there would be a total of ~3ng (on average) for a part sample in a well, and assuming a suspension with 10ul, there would be more than enough for PCR reactions. However, transformations are recommended over PCRing directly from the kit, because you can now store that part long term in glycerol form and you can prepare more of that part easily.

So the reason to prefer transformation over PCR seems to be long-term storage and future amplification.

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