ligated vs. supercoiled - (May/08/2007 )
I'm reading several protocols about cloning, transformation etc.
All of them indicate the "supercoiled plasmid" amount to use, except one that reports two different amounts of DNA (supercoiled and ligated plasmid).
Now, I know the basic definitions of these two terms but I really don't know - in practice - if I will work with supercoiled plamisd.
When you have a supercoiled plasmid? How can I understand which configuration is mine???
Thanks very much!
My understanding is usually the plasmid DNA itself will form supercoil conformation. What kinda of application are you dealing with your plasmid? Transformation? I dont think it will make any difference. But i might be wrong. Still new.
First I will make the cloning in E. coli then, after miniprep, Agrobacterium electroporation.
I really don't know if it makes differences, I'm still trying to understand...still new too...
You force the opening of small pores in the membrane of Agro to allow the entrance of DNA, so a supercoiled form - which is more compact - is more advantaged than to more relaxed ones in entering inside the bacteria.
I thought it made sense...
I will tell you my experience. Plasmids have different conformations. Either it is supercoiled, half coiled, relaxed state or linear form. I just transform the plasmid into E.coli cells. And i guess this applies either you are using Agrobacterium or yeast. Maybe you can try it out and check how efficient is the transformation?
Thanks again Timjim and congratulations for your thesis!
Oh.. thanks. Everyone seems to know that I am done with my thesis. So happy.
isnt it that supercoiled and ligated plasmid in the context of the question means supercoiled= uncut plasmid, which means ordinary transformation with the competent cells that can be usually done thru a simple heat shock or electroporation , and that ligated plasmid= plasmid that had undergone ligation step, then followed by transformation??
transformation with uncut plasmids is way easier as compared to transformation with ligated plasmid since u dont have idea how much plasmid and inserted was ligated.
I think that supercolied plasmids are smaller in size and easier to get into cells then long ligated plasmids. this is why you get higher transformation efficiencies with supercoiled plasmids.
If you use your plasmid right after ligation, then it is linear and the transformation efficiency is lower and depends on the efficiency of ligation.
If you first transform your ligation into cells and grow these then do a prep. then your plasmid becomes supercoiled and has a higher transformation efficiency.
are you using your plasmid from a ligation or DNA prep?
Now everything is much more clear!!!