This chapter describes the methods used to prepare cDNA and genomic libraries from which it has isolated clones for fibronectin and for its receptor in fibroblasts. This chapter have used both the DNA polymerase I-S1 nuclease and the RNase H-DNA polymerase I protocols to prepare double-stranded cDNA. Both have been optimized to give the longest cDNA attainable. Both work but the RNase H protocol is somewhat simpler and may give clones extending closer to the 5' end of the mRNAs. In particular, it was emphasizes on different methods of cDNA cloning based on screening with antibodies using the prokaryotic expression vector λgtl. This chapter also discusses several recent improvements in cDNA and genomic cloning protocols. This chapter also describes various methods that have used for the preparation and analysis of fusion proteins containing segments of fibronectin, which can be produced in large amounts in bacteria for use as immunogens or in functional studies. The methods are general and could be used for other extracellular matrix proteins and indeed for any other proteins.
All Science Journal Classification (ASJC) codes
- Molecular Biology