CLONING

General Method for Sub cloning:

1. Choose an appropriate vector (e.g., T7, pL, lac, tac) taking into account the presence of convenient sites by restriction enzymes.

2. isolate both the insert encoding your protein of interest and the vector upon digestion with appropriate restriction enzyme.

3. Transform a competent bacterial host (e.g., E. Coli) using standard protocols and select transformants on the basis of resistance to appropriate durgs.

4. Measure overexpression of the cloned gene product by

    a) standard gel electrophoresis of the whole cell extract.

    b) immunoblotting (e.g., use an antibody raised against your protein of interest.

    c) a biochemical assay of the known activity of the protein

Cloning of cDNA:

Restriction enzymes can be used to clone complementary DNA (cDNA). (1) First,  a poly-T primer (used because most mRNAs have a poly A tail) is hybridized to mRNA and then transcribing the mRNA into cDNA with the enzyme reverse transcriptase. (2) the resulting mRNA-cDNA hybrid is then turned into dsDNA by treating the hybrid with an alkali which destroys the RNA but not the DNA strand. The resulting sscDNA forms a small hairpin at its 3' end, which serves as a primer for synthesis by the enzyme DNA of a complementary DNA strand. (3) After synthesis, the hairpin is cleaved with S1 nuclease to generate the dsCDNA. (4) the cDNA can now be cloned into a plasmid vector and then the vector can be transformed into E.coli. A collection of DNA sequences within plasmid vectors representing all the mRNA sequences derived from a cell or tissue is called a cDNA library. Such a cDNA library can be screened for genes of interest as for example, by using southern blotting.

Cloning of DNA:

Genomic DNA on chromosomes can be cloned using the following general procedure. (1) The chromosomal DNA is cleaved with restriction enzymes that produce sticky ends and then that DNA is cloned using a virus vector like bacteriophage ג. In the alternative, the genomic DNA can be cut with an enzyme that produces a blunt end, short linkers can be added to that blunt end enabling that end to thereafter be annealed to the sticky end of the vector.

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