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Need information about pBR332


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#1 Trogie

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Posted 12 December 2009 - 01:35 PM

Hi,

I have to give a presentation and as subject I chose pBR332. So far I havent found that much about it so I was wondering if any of you could provide me with a little extra information.

The main things I want to know more about are:
- the origin: thus a little information on how Bolivar and Rodriguez actually created it
- how to use it: like detection once sequence is inserted and stuff
- an example of how it is used currently or why it wouldnt be used

Anyway thanks for the help already.

#2 pDNA

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Posted 13 December 2009 - 12:59 AM

try this powerpoint presentation! ...this should have all the informations included you are looking for!

Next time try to use google first ...i took me 5s to find this piece of work!

Regards,
p

Attached Files

  • Attached File  Anu.ppt   330KB   820 downloads


#3 Trogie

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Posted 13 December 2009 - 03:57 AM

try this powerpoint presentation! ...this should have all the informations included you are looking for!

Next time try to use google first ...i took me 5s to find this piece of work!

Regards,
p


Thnx a lot, I tried google before but apperently didnt use the right command as mostly I got all useless stuff. Just wondering, what command did you use?

I also read through the ppt and got indeed most of the answers. All I just want to know now is how it's used. I know how to insert DNA and such but I dont know how you'd select the successful transformations and stuff. Also is this used to produce proteins and later extract them? If so, how would that be done?

As you may have noticed I dont know much of this. Basically because we'll see this kind of things only next year or even later... I'll continue to search on my own too. I suppose the information I still need is also similar for other cloning vectors.

#4 pDNA

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Posted 14 December 2009 - 03:46 AM

you can select for pBR322 containing clones by using the antibiotics Ampicillin or Tetracycline (the resistance genes are both encoded on the plasmid). So you will do a transformation of your E. coli host with pBR322, either using electroporation or heat-shock, and then plate the cells on a agar plate with one of the two antibiotics. Only pBR322-carrying cells are going to survive in this step.

pBR322 can be used for the expression of proteins if you will insert a gene together with a promoter (there is no promoter on the plasmid that will drive the expression of your gene if you only will insert the coding region in the multiple cloning site). pBR322 is a low copy number plasmids (this means there are only ~20 copies of this plasmid inside the cell) ...for protein production normally high copy number plasmids are used, like the pET-series from Novagen. The have a T7 promoter that can be used for expression together with strains that encode for the T7 RNA polymerase. This would be an example for a very strong expression system, yielding large amounts of protein. If you want to learn more about that system look it up here:
http://bioenergy.asu...periment-I.html

Here you have a detailed protocol on the expression and purification of a protein using the T7 system.

Hope this will help you get along!

Regards,
p

#5 HomeBrew

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Posted 14 December 2009 - 06:31 AM

Because you're doing a presentation, let me offer some small clarifications for accuracy, in case they weren't already clear:

you can select for pBR322 containing clones by using the antibiotics Ampicillin or Tetracycline...

Presuming your recipient cells are sensitive to the antibiotic you choose, and that you haven't cloned your insert into one of these genes, thus disrupting it.

So you will do a transformation of your E. coli host with pBR322, either using electroporation or heat-shock...

There are other ways to get pBR322 into cells (e.g. transduction), so this is not an exhaustive list. Also, "heat-shock" is shorthand for "transformation of chemically competent cells", of which one step of the protocol involves incubation of the cells for a short period of time at high temperature. Simply heat shocking the recipient cells in the presence of the plasmid will not result in transformation.




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