cDNA cloning question - (Sep/27/2005 )
I want to make a cDNA clone of a specific protein expressed in abundance in a specific cell type. Assuming I have made cDNA clones of all the mRNAs produced in that cell type, how do I find the specific protein I want? I don't have a probe and don't know the sequence. Is there a way of finding which cDNA is most abundant maybe?
the only thing you know is that you have a cDNA library? I think you will need more information to go on
do you have a cDNA sequence of the protein you seek? Do you have the sequence of any of its homologues in other species? has your species been entirely cloned? you could make some predictions if you have any of this information
do you know anything at all about the protein you are looking for?
O.k I now know that my protein is very similar to a protein from another species. However I only have cloned fragments of this similar protein and no way of getting more. Could I label all the fragments, denature these 'probes' as well as my newly synthesised cDNA and then screen to see if the fragments all join up to one of my cDNA molecules from my library? If that was successful how do I isolate the cDNA of my protein?
you have to transform the cDNA library and get colonies of bacteria that only contain one plasmid then you could do a colony lift and use your probes and you get the plasmid back by going back to your original plate and growing up the positive colony that bound to the probe....
you could plaque hybridization if your cDNA library is in a phage
there are some protocols on POL home
I was going to also suggest plating your cDNA library at dilution, and doing colony lifts on Whatman filter circles and trying to hybridize your probe to the filters. We've used this technique to fish out clones from a genomic DNA library.
You must, of course, be sure a homolog of your gene does not reside on the chromosome of the host cells (those containing your cDNA clones), or this won't work. If your host cells do contain a chromosomal copy of the gene, you'd need to do plasmid extractions on colonies from your library (perhaps in a 96-well format) and do a Southern blot of the plasmid DNA run out on a gel.
If you have plasmid DNA of your library, you might also consider degenerate PCR to find a postive clone...
Thanks so much! You've all been really helpful and I now have my cDNA
do a microarray?