How can i clone a plasmid?

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Karen A. Darbinyan

Hello see below info, may be helpful for you.

https://www.ncbi.nlm.nih.gov/books/NBK21498/

https://www.addgene.org/protocols/pcr-cloning/

Good luck!

Shadi Zerehpoosh

Karen A. Darbinyan Thanks a lot

Your welcome !

Abeeb Abiodun Yekeen

@ Shadi Zerehpoosh

Hi,

It might be appropriate to first ask you a pertinent question;

Why would you want to clone (if that means transforming or "producing more copies of") a plasmid you know nothing about?

Probably to have enough quantity of it for downstream study/analysis?

However, to answer your question, since you have no knowledge regarding the said plasmid, you might want to transform it and plate on a number of solid media each with a different antibiotic selection (consider the more common antibiotics first). This would at least provide you with information as regards its resistance and you would have transformants from which you can re-extract the plasmid.

Hi dear Abeeb .@ Abeeb Abiodun Yekeen

Thank you for answering.

yes, i want to producing more copy of this plasmid to reach enough quantity to do more study on it.

i tried it with two antibiotics till now.( Ampicillin and Kanamycin )but it doesn't worked.which other antibiotics would you offer to try???

Hi dear Abeeb

You are welcome.

You can try Tetracycline, Streptomycin, Chloramphenicol, Spectinomycin. These are pretty common in many plasmids...

Download the attached for a list of some antibiotics and their recommended working concentrations...

Thanks a lot...

Michael J. Benedik

Why don't you tell us a bit about the origins of the plasmid. I would note that if this is a plasmid from a natural strain, it might not be antibiotic resistant. Most natural plasmids are not, that is an artifact of how we work in the laboratory.

Alireza Mordadi

There is a big question mark!!!

When you do not know anything about your plasmid, even you cloned it, how host cells gonna keep the plasmid, how do you want to use it,.... and many more and more questions.

Alireza Mordadi Hi.how are you? Thanks for answering ...

Let me enlighten you. I have a plasmid which i don't know any thing about it's backbone.i know the gene that inserted on it.and i want to clone it to reach enough quantity to do more study on that gene.

But I got into trouble in first step...

Hi Mr Michael J. Benedik .and thanks a lot for answering my question...

In fact, this plasmid is a synthetic plasmid made in a recombinant form. and it contain a gene... I guess it should have an antibiotic resistance gene in it...

A question was created after reading your message. How can we clone a plasmid that has no antibiotic resistance gene?

isn't it necessary for cloning of a plasmid to have at least one extra feature to give benefit to it's host cell???

If you know your gene, design proper primers and do PCR then send it for sequencing to be certain that your sequence is right. So you can clone it on proper vector and right host that you need for further study.

Take it, the main point of cloning is get information about a gene, such as function, expression, products and etc.

But about your question for benefit, you are right, the main reason that horizontal gene transform happened in nature, like transfer a plasmid or a gene on plasmid is be beneficial for host.

But if you want to study a gene throw cloning, you have to have some options such antibiotic resistance gene to prove you cloned your plasmid.

Assuming the plasmid is for use in E. coli, then it is highly likely to carry resistance to one of the four antibiotics, ampicillin, kanamycin, tetracycline or chloramphenicol. While there are some plasmids carrying other markers, they are pretty rare. On the other hand, if the plasmid is a shuttle plasmid for a different organism then it might be something different.

Hi...

Thanks a lot .it was such a useful guidance...

Hi....thanks a lot