I want to express an enzyme in host bacteria (E. coli), but I need to make sure the copy number of the plasmid in a single host cell.
You could run a real time PCR with probing for at least 2 targets. 1 of them will be probing for the plasmid of your interest; the other(s) should probe a bacterial chromosome gene with only 1 copy to use as DNA standard. By examine the number of plasmids/ number of copies of bacterial gene. One can deduce the number of target plasmid/ host cell.
if your DNA is labeled then after transfer and determination of radioactivity?
When you know the size of the plasmid and the yield from a miniprep and also know how dense the culture was, then you can also make an estimation about it.
You could run a real time PCR with probing for at least 2 targets. 1 of them will be probing for the plasmid of your interest; the other(s) should probe a bacterial chromosome gene with only 1 copy to use as DNA standard. By examine the number of plasmids/ number of copies of bacterial gene. One can deduce the number of target plasmid/ host cell.
Hi
If you know the origin of replication of the plasmid used that will help you to know the copy number.
This link may help you
http://www.qiagen.com/knowledge-and-support/faq/?ID=1f42840e-fbd7-4734-b0cd-e17372a9e5a4
Gia-Phong and Lakhal have it right. An example of a protocol is also given in Assessment of the Mobilizable Vector Plasmids pSUP202
and pSUP404.2 as Genetic Tools for the Predatory Bacterium
Bdellovibrio bacteriovorus (DOI 10.1007/s00284-010-9748-5)
Dear all,
i am very much in favor of Christians approach, its so easy and old-school. Also, the link given by Abderrahmane Bengrine is useful as the choice of ori has a huge impact on the copy number. Q-PCR based approaches are less accurate and more tedious. Keep it simple!
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