8 replies to this topic

[Nortagig]

Hi,

can E. coli cells be transformed with digested plasmid?
Whatever is the answer, what is the reason?
Thank you very much

Edited by Nortagig, 03 February 2010 - 09:28 PM.

[microgirl]

I'm not sure I really understand your question. I'm sure you can get your digested plasmid into your cells, but I don't know why you'd want to get it in in a digested state since it probably won't do what you want it to once it's in there.

Nortagig, on Feb 3 2010, 10:13 PM, said:

Hi,

can E. coli cells be transformed with digested plasmid?
Whatever is the answer, what is the reason?
Thank you very much

[Nortagig]

Thank you for replying,

I did not say I want to get a digested plasmid into the cells, is just for knowledge. You say you are sure the digested plasmid can get into the cell, but, as a circular plasmid does, would a digested plasmid confer antibiotic-resistance?
Thanks



I'm not sure I really understand your question. I'm sure you can get your digested plasmid into your cells, but I don't know why you'd want to get it in in a digested state since it probably won't do what you want it to once it's in there.

[fishdoc]

Nortagig, on Feb 4 2010, 08:37 AM, said:

Thank you for replying,

I did not say I want to get a digested plasmid into the cells, is just for knowledge. You say you are sure the digested plasmid can get into the cell, but, as a circular plasmid does, would a digested plasmid confer antibiotic-resistance?
Thanks

In some bacterial species, you can electroporate a linear piece of DNA, and if it contains homologous DNA, it can integrate into the genome. This integration would allow for expression of any genes encoded on the DNA, including antibiotic resistance genes. But because it's integrated, it really isn't a self-replicating DNA. There are linear plasmids out there. I don't know that any are used for cloning purposes or anything like that, as I'm not too familiar with them.

[tfitzwater]

Years ago I transformed gel-purified HinD III-cut plasmid into E. coli by the CaCl2 method and got lots of colonies.  Treating the linearized DNA with calf alkaline phosphatase prior to transformaton eliminated the colonies.

[microgirl]

Your problem would be getting the cut plasmid to replicate and have all the genes on it work properly. I guess if it was cut in the right place it might still work - there are linear plasmids out there.

(I did not say I want to get a digested plasmid into the cells, is just for knowledge. You say you are sure the digested plasmid can get into the cell, but, as a circular plasmid does, would a digested plasmid confer antibiotic-resistance?
Thanks )

[Nortagig]

Usually when you set up a transformation you need to use a control vector. That vector is the same you used for ligation but without any insert. My question is, is that digested vector able to generate antibiotic-resistant colonies?

Edited by Nortagig, 04 February 2010 - 10:47 AM.

[fishdoc]

Nortagig, on Feb 4 2010, 12:47 PM, said:

Usually when you set up a transformation you need to use a control vector. That vector is the same you used for ligation but without any insert. My question is, is that digested vector able to generate antibiotic-resistant colonies?

Probably not. You would need to use uncut vector or recircularize the vector before transformation.

[tfitzwater]

My experiment appeared to indicate that the bacteria was ligating the linear plasmid, since CIP-treatment eliminated the effect.  This was a pBR322 vector selected for amp and tet resistance.  All of the proper controls were run.  The colonies came up on my no ligase control plates.