1. making suspension cultures which pool many colonies from the
2. removing plasmids and binding them to nitrocellulose,
3. denaturing the bound plasmids and hybridizing with total cellular mRNA,
4. eluting bound mRNAs from each filter and transcribing their proteins in vitro,
5. screening the proteins to identify the colonies expressing the gene of interest,
6. rescreening to identify the single clone expressing the gene.
A much easier technique involves detecting the protein of interest while still within the living E. coli cells. In order for this to work, however, human cDNA must be cloned into special expression vectors which overcome difference in regulatory mechanisms in each species:
1. Expression vector pUC19
2. The cells containing the expression vector are
a. cultured on plates, then portions of colonies are transferred to nitrocellulose
b. lysed and incubated with antibodies specific for the protein to be screened
c. incubated again with a radioactively-labeled protein which is also specific for the
antibody (such as Staph A protein labeled with 125I).
d. nitrocelluose sheets are washed, then sandwiched between plastic wrap and
used to expose x-ray film. Example
3. After exposure, the film will show dark spots indicating the colonies expressing the
protein. Lining the film up with the original pate enables the researcher to separate
and isolate the cloned cells.