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Generate Supercoil Plasmid


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5 replies to this topic

#1 Adrian K

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Posted 15 August 2010 - 03:11 AM

Hi All,
I just came across of this thought when I'm having an afternoon nap.
I know is possible for us to generate a linearized plasmid by doing inverse PCR.
From here, is it possible if we can make it in a form of supercoil?
Any idea?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#2 phage434

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Posted 15 August 2010 - 04:45 AM

PCR fragments can be ligated to form circular plasmids, which can be transformed. In the cell, topoisomerases will supercoil plasmids and maintain the proper level of coiling. The final ligation of virtually every cloning experiment ligates a linear to a circular fragment, which is then transformed. PCR fragments are no different than other DNA (modulo possible methylation).

#3 Adrian K

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Posted 15 August 2010 - 06:29 AM

in order to make supercoil plasmids synthetically without using a living host, not possible?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#4 phage434

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Posted 15 August 2010 - 08:21 AM

DNA gyrase will introduce negative supercoiling in buffer + ATP. See NEB product M0306. This will only work on un-nicked circular DNA fragments. Supercoiling can be reversed with Topoisomerase I.

#5 Mycobacterial Madman

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Posted 15 August 2010 - 06:23 PM

why would you want to do this? for more robust plasmid storage or something?

if you're after better transformation efficiency, I remember reading that closed circular plasmid DNA transforms E. coli with higher efficiency than supercoiled plasmid DNA...

#6 Adrian K

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Posted 16 August 2010 - 06:57 AM

Hi phage434,
Thanks for your information and prompt reply.

Mycobacterial Madman: I was told supercoiled plasmid is good quality plasmid... that's the reason I'm looking into that...
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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