Clone blunt ended insulin gene into pbr322
Posted 19 October 2004 - 05:39 PM
Posted 20 October 2004 - 12:44 AM
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
Posted 20 October 2004 - 05:11 PM
Posted 21 October 2004 - 05:55 AM
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!