How many cDNA clones does one have to screen to assure that one will find their

Question

How many cDNA clones does one have to screen to assure that one will find their cDNA of interest? What vector(s) would be most appropriate for use in construction of a cDNA library? Assume one has not been lucky enough to isolate a full length cDNA clone. What part of the coding sequence would be missing -that coding for the amino terminal or carboxy terminal of the protein? If one were going to use a heterologous probe for library screening, what conditions of temperature of hybridization and salt concentration might one select?

Explanation / Answer

High density screening enables the actual experimentalist to screen involving 100,000 as well as 1,000,000 independent plaques on a single plate and it is possible to screen for a particular cDNA, which can present one copy per cell. Screening is performed by replica plating procedure. Following phage infect E.coli and sort individual plaques, an ideal spatial representation on the attacked plaque can be produced by setting nitrocellulose on the surface of the E-coli lawn. Nitrocellulose binds DNA together with great avidity and thus some of the DNA of each plaque may be transferred to nitrocellulose paper or it can perhaps several different nitrocellulose papers. The DNA from the actual library will then be cross-linked to the filter and protein can be washed off. The plaques of cDNA of interest can then be screened using hybridization assay.

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