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Can iGEM distribution parts be directly PCR'd?
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Kevin Kolbe
Can iGEM distribution parts be directly PCR'd?
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.
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.
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.
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.
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.More
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.More
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?More
Here is the feedback I got from iGEM HQ:
So the reason to prefer transformation over PCR seems to be long-term storage and future amplification.
Here is the feedback I got from iGEM HQ:
So the reason to prefer transformation over PCR seems to be long-term storage and future amplification.
More
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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.
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.
More
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