|MadSci Network: Molecular Biology|
DNA cloning is one of these fields where one can "cook" approximately what he wants. It all depends what you want to do with your plasmid afterwards, and what is your source of material.
Either you want the gene itself, with all the introns. In this case I would suggest you buy a BAC clone (easily found on NCBI). You can also PCR the genomic DNA, but in this case, good luck (quite difficult).
I think you rather want the expressed sequence (peptide equivalent), and in this case you start with mRNA, cDNA, or an already existing plasmid. You want to put it into a plasmid and transform bacteria and/or transfect cells. You have the choice :
- you find (2 different) restriction enzymes that cut near your gene and not in your gene. The only reason you want to cut as close as possible to your gene is that longers insert have weaker cloning efficiencies, make plasmids that are less replicated by the bacteria and more likely to be rejected by eucaryotic cells.
- you PCR the cDNA/DNA with primers in your start and stop codons, or primers that are in the UTRs (untranslated regions). There should be one in 3' and one in 5'. Then you directly clone in a TOPO vector (see http://www.invitrogen.com/content.cfm?pageid=4073&CID=KNC- GOOGLE&s_kwcid=topo%20cloning|529970173 or search Invitrogen for TOPO), and then cut with the restriction enzymes of your choice.
- you PCR (at the worst case nested PCR) with tailed primers. They have a 5' extension that is not specific of your gene but that contain restriction sites. You can design them without any space between your restriction site and your ATG. After the PCR you cut and clone. That is what I would do...
- Last option, weirdly tricky but usefull : you can synthetize your gene de novo. See Stemmer et al. in Gene, 164 (1995) 49-53... This allows you to easily change amino acids, or optimize codon usage, for example.
For protein expression, do not forget to check your frame (common beginner mistake)
Contact me through MadSci (reply to the emailed answer) if needed!
Hope it helps
Diverti tu bene
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