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How to make an empty vector (such as pET ecoli vector) using plasmid miniprep an

microbiology

Best Answer OldCloner, 23 July 2019 - 11:15 AM

Hi tbd,

 

There is no difference in making plasmid minipreps of empty vectors and in making plasmid minipreps of vectors with inserts. You just need to know what antibiotic resistance gene is carried by the vector, so when you grow your bacterial culture, you select for cells that contain the plasmid (empty or not).

 

However, I’m a bit confused about what your starting material is.  Do your BL21 cells already contain the pET vector?  If so they are no longer competent, they are just frozen cells containing plasmid. It is best to plate a loopfull of the frozen material on selective agar for single colonies first, then pick a nice healthy- looking, well separated colony, and use it as inoculum for the next step. And then you would grow them in the appropriate antibiotic broth and do the miniprep according to instructions.

 

 However, If you have been given purified pET PLASMID DNA (not in cells) and you need to make more by putting it into BL21 competent cells first, you need to perform a step called transformation to get the plasmid into the competent cells.  You will need to look up the procedure for transforming that strain of E. coli. If you have frozen COMPETENT cells, there is a procedure for thawing an aliquot, adding the plasmid DNA, then doing a treatment to make the plasmid enter the cells (usually a heat shock, unless you are using “electrocompetent” cells and electroporation), then after following all the instructions, you plate the cells on agar containing your selective antibiotic and incubate them.  Next day look for surviving colonies and they should now contain the plasmid.  Then you can pick a colony and grow it up in broth to make the plasmid prep.

 

Note: you generally need only a few nanograms of plasmid DNA to transform cells (lots less than you would use if you were transforming a LIGATION mixture.)

 

If your frozen BL21 cell stocks are just frozen cells WITHOUT vector, and not competent, then there is a procedure for making them competent. But for most people these days, it is a lot easier to buy stocks of frozen competent cells.

 

Hope this makes sense to you! Good luck!

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#1 tbd

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Posted 22 July 2019 - 06:44 PM

Basic idea: I need to isolate an EMPTY pET vector from ecoli genomic DNA via plasmid miniprep, but miniprep procedures require you to have colonies that you then incubate over night. Currently, I know how to do this with plasmids with gene inserts in them, but I need to figure out how to grow cells with just empty plasmids. This is probably such a dumb questions, so thanks for the help haha

 

My idea currently is to perform plasmid miniprep on BL21 competent cells (which apparently my pET vector comes from)..

1. I would use a sterile scoop and dip it in BL21 frozen stock of ecoli competent cells and swirl this into a LB and antibiotic mixture. Then I would incubate overnight

2. I would perform plasmid miniprep: pellet the cells, add buffer, lyse the cells, etc.

 

Does this sound about right?

Any help would be greatly appreciated, and I will stick around as much as possible to answer any clarification questions needed!


Edited by tbd, 22 July 2019 - 07:17 PM.


#2 OldCloner

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Posted 23 July 2019 - 11:15 AM   Best Answer

Hi tbd,

 

There is no difference in making plasmid minipreps of empty vectors and in making plasmid minipreps of vectors with inserts. You just need to know what antibiotic resistance gene is carried by the vector, so when you grow your bacterial culture, you select for cells that contain the plasmid (empty or not).

 

However, I’m a bit confused about what your starting material is.  Do your BL21 cells already contain the pET vector?  If so they are no longer competent, they are just frozen cells containing plasmid. It is best to plate a loopfull of the frozen material on selective agar for single colonies first, then pick a nice healthy- looking, well separated colony, and use it as inoculum for the next step. And then you would grow them in the appropriate antibiotic broth and do the miniprep according to instructions.

 

 However, If you have been given purified pET PLASMID DNA (not in cells) and you need to make more by putting it into BL21 competent cells first, you need to perform a step called transformation to get the plasmid into the competent cells.  You will need to look up the procedure for transforming that strain of E. coli. If you have frozen COMPETENT cells, there is a procedure for thawing an aliquot, adding the plasmid DNA, then doing a treatment to make the plasmid enter the cells (usually a heat shock, unless you are using “electrocompetent” cells and electroporation), then after following all the instructions, you plate the cells on agar containing your selective antibiotic and incubate them.  Next day look for surviving colonies and they should now contain the plasmid.  Then you can pick a colony and grow it up in broth to make the plasmid prep.

 

Note: you generally need only a few nanograms of plasmid DNA to transform cells (lots less than you would use if you were transforming a LIGATION mixture.)

 

If your frozen BL21 cell stocks are just frozen cells WITHOUT vector, and not competent, then there is a procedure for making them competent. But for most people these days, it is a lot easier to buy stocks of frozen competent cells.

 

Hope this makes sense to you! Good luck!


Edited by OldCloner, 23 July 2019 - 11:21 AM.

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