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Clone blunt ended insulin gene into pbr322


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#1 Akasha

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Posted 19 October 2004 - 05:39 PM

Okay, I am a real new microbiologist. In fact, I am currently doing my very first microbiology course in college. We are talking about Recombinant DNA technology and I'm like :huh:. Can anyone tell me what restriction endonuclease to use if the vector I am using is pbr322 and the gene I want to clone is a blunt ended insulin? Help! I need to finish writing up a paper about this and I cannot find the info online or in my dum textbook.

#2 jadefalcon

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Posted 20 October 2004 - 12:44 AM

pBR332 is kind of a standard vector. you first have to tell what you want to do, do you want to express your gene in this vector, then you have to be cautious if the insertion is in frame and near the promotor of this plasmid.
if you just want to clone your blunt end gene (as is generated by PCR) into the vector, you'll need a blunt ended vector. so a restriction digest with any blunt end cutting enzyme would do. preferably you use an enzyme, that is a unique cutter, i.e. the cuts only once in your vector.

for pBR332 EcoRV would do, for example.

so, in a step by step manner:

1. get your blunt end gene
2. restriction digest your vector with EcoRV
3. phosphorylate your PCR product
4. dephosphorylate your vector
5. ligate the two
6. transform into competent E.coli & put those on selevtive media
7. pick single colonies & do small volume cultures
8. extract plasmid & check by restiction digest or PCR for insertion of your gene

mike
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#3 Akasha

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Posted 20 October 2004 - 05:11 PM

Hey jadefalcon. Thanks for your speedy reply. The thing is, I need to use a restriction endonuclease that cuts pBR322 at one of the antibiotic resistance sites. Searching the web, I found that Hae3 will do that. However, I am not sure about how reliable my internet source is. What do you think? And do you know which antibiotic resistance site this cuts?Thanks again for your help. :huh:

#4 jadefalcon

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Posted 21 October 2004 - 05:55 AM

ok, you first have to come up with the plasimd sequence or a detailed vector map.

the sequence is available at the NCBI database (http://www.ncbi.nlm.nih.gov) accession number J01749.

there you find additional information about where the "features" of this plasmid are located in the sequence (e.g. tet resistance gene from 86-1276, bla from 3293 to 4153).

so, now you know where the areas you have to cut are located, you are searching for a restriction endonuclease, that will leave blunt ends after digestion, cuts preferably only once in the whole vector and has a recognition site in the area of your interest. so stuff the sequence in a computer-programme that can show you the restriction sites in the sequence. pick one that fits all of the above reqirements- there you are!

in your case: EcoRV at 187, NruI cuts at 976, ScaI at 3846. that's about it. I personally would prefer EcoRV above the others, since it's a standard enzyme, reliable, highly available.

the enzyme you mentioned, HaeIII will in fact cut in one of the restriction genes, you're right there,

BUT (big BUT)

HaeIII has a very short recogition sequence (GGCC) that appears 22 times in the pBR332 sequence. So, if you'd do a restiction digest of the plasmid with that enzyme, you'd end up with very many very little pieces of DNA that won't do anything anymore. in other words, a digest with HaeIII would destroy your vector! ;)
so, personally not recommended! :o

mike
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