making recombinant protein
Posted 02 December 2011 - 12:43 PM
I have the sequence of the two fragments, and I have genomic DNA as the template.
But I don't know how I can make the recombinant protein.
Would you please help me, let me know the methods, or if there are references?
Thanks a lot!
- Lpymly254 likes this
Posted 02 December 2011 - 01:23 PM
- stitch the two genes by PCR ...details see [url="http://openwetware.org/wiki/Sauer:Stitching_Genes_by_PCR"]here[/url].
- clone them into an expression vector (e.g. pET vectors)
- transform your expression plasmid into e.g. BL21(DE3)
- do the expression according to the [url="http://www.google.at/url?sa=t&rct=j&q=pet%20manual&source=web&cd=1&ved=0CCgQFjAA&url=http%3A%2F%2Flabs.fhcrc.org%2Fhahn%2FMethods%2Fbiochem_meth%2Fpet.pdf&ei=sEHZToP2IMzGtAbL6PjLCw&usg=AFQjCNFV2qS31yBoUrU_WY03IA7SeLnIoQ&cad=rja"]pET manual [/url]
Posted 09 December 2011 - 06:32 AM
I like your stiching genes by PCR. Thanks! I will need it. I am running degenerate PCRs on 2 libraries: genome walker (with Clonetech) and marathon cDNA made by previous workers. I found some sequences of the gene of interest. I am planning to do complementation and express it in yeast BUT:
I don't know how far should I walk? At the moment I have total sequences of 3kb from the genome walk which probably includes introns
I have very little cDNA to work with so I try to be careful in each step
What should I do to get the gene expression work? and
Walk further both directions? Stich some pieces and try the expression?
Make some primers and get the cDNA sequences to express only the cDNA (I may loose some cDNA if my primers fall in intron region-- which I don't know from where to where...)
And I would like to start with Gateway.
Any advices are welcome.