I want to design construct on my selected vector.However how do I know whether my plasmid is high or low copy number?
You can check the plasmid's origin of replication.
http://blog.addgene.org/plasmid-101-origin-of-replication
You can check the plasmid's origin of replication.
http://blog.addgene.org/plasmid-101-origin-of-replication
https://www.qiagen.com/no/resources/faq?id=1f42840e-fbd7-4734-b0cd-e17372a9e5a4&lang=en
Check the ori of the plasmid. And,Ii there are centromeric sequences as in some off the yeast vectors, they are low copy.
- The copy number of a plasmid is depending on the origin of replication (ori) they contain.
- The different oris regulate the replication mechanism of the plasmid. High-copy plasmids often contain mutated versions of an ori.
1. when you growth the selected vector, you will know whether it is high copy or not.
If the plasmid is not temperature sensitive, when growth 5ml LB in 37 degree ovn
, using normal miniprep kit, you should get 25-30ul plasmid DNA with concentration about 1ug/ul
(Then this vector is high copy number.)
2. If you can found the plasmid in addgene, check the section: Growth in Bacteria
It will tell you Copy number
3. In theory checking the replication origin is the correct answer.
But you should be known that for some of the ori, mutations can change the copy number.
For example pBR322 is a low copy number plasmid with a ori called pMB1.
However, other plasmids with pMB1 ori could be high copy number.
For exmple: pUC vectors and pTZ vectors are high copy number.
Indeed, the pUC and pTZ have a mutant version of pMB1.
So make sure you really understand that is the copy number of its parental plamid is important.
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