gene cloning, where to start? - (Jun/26/2013 )
I want to clone mouse IL-13 gene which is about 3.7 Kbp into a P-Live plasmid. I don't know where to start. I thought about two ways:
1- finding its mRNA sequence (From where?) / designing primer for it/ makings cDNA for it/ amplifying the DNA/ cloning in the plasmid
2- finding its gene sequence/ designing primer for it/ amplifying the gene of interest/ cloning in the plasmid
What is your idea? Is there any book or website that tells me what to do step by step?
A good source book for protocols is Sambrook, Fritsch and Maniatis, Molecular Cloning: a laboratory manual.
These sorts of things are very commonplace in the lab - it is quite likely that IL13 will have already been cloned by someone - you can try looking on Addgene (addgene.org) for plasmids containing it.
For the cloning do you want introns and exons or just the coding sequence? Most people only want the coding sequence so that they can express it in a system and then study the effects - this is done off mRNA and cDNA followed by PCR usually. mRNA sequences can be found on the NCBI website under the nucleotide option.