Did sequences affected amount of supercoil? Regardless of extraction in low temperature other factors can be handle for earn more supercoil? I have several plasmid that some of them ( such as puc57 ) in different condition even room temperather or higher, give considerable amount of supercoil but others (such as egfp.n1 ) even in 4°¢ didn't give superciol or have a little amount and isn't repeatable. I use favorgen minikit for plasmid extraction and my institute don't have ultracentrifuge.
thanks
Hello, The topoisomerases, enzymes that modulate the degree of DNA supercoiling, are divided to two families: typeIenzymes transiently cleave and reseal one strand of duplex DNA and type II enzymes cleave and religate both DNA strands. These two families are divided further into subfamilies, which can be distinguished on the basis of protein architecture, DNA substrate preference, reaction outcomes (net loss or gain of supercoils; complete or partial supercoil removal), and requirements for metals and ATP. The catalytic mechanism and activity for each family of topoisomerase enzymes are different and and depends which activity and mechanism is predominant the same plasmid in same condition can give you different amount of supercoil.
Hope this info will help you.
Good luck!
Hi karen
Thank you for complete explanation.
But I use same host (dh5 alpha) and same media (LB) in each plasmid extraction.
Did you mean host changing can be affective?
Any changes in host in any conditions can cause changes. I think supercoiling process cent be strictly controlled due to many variable parameters.
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