I. Expression and purification of recombinant Alkaline Phosphatase (AP)

• A. Grow E. coli strain carrying pProPhoA and induce protein expression
• B. Prepare cell extract using sonication
• C. Detect AP by Western blot (immunoblot) using anti-bacterial AP
• D. Purify recombinant AP (His-tagged) by affinity chromatography

II. Analysis of AP protein purification

• A. Quantitate protein by colorimetric assay
• B. Visualize proteins by SDS polyacrylamide gel electrophoresis
• C. Measure AP activity by enzymatic assays.

III. Recombinant DNA Technology: Generate a recombinant DNA clone to express bacterial AP in E.coli

• A. Restriction enzyme digestion of AP gene plasmid and expression vector
• B. Analysis of digests by agarose gel electrophoresis
• C. Purification of DNA fragments
• D. Ligation and transformation into E. coli
• E. Isolation of plasmid DNA from transformants (minipreps)
• F. Restriction enzyme analysis to identify correct plasmid construct ("pProPhoA")

IV. DNA sequence analysis

• A. Sequence pProPhoA by dideoxy sequencing
• B. Analyze results using phosphoimager and autoradiography
• C. Search DNA and protein databases for human AP homologues

V. Polymerase Chain Reaction

• A. Use PCR to amplify human intestinal AP from human genomic DNA
• B. Analyze PCR products by restriction digestion and gel electrophoresis

Vl. Southern Blot Analysis

• A. Restriction enzyme digestion of human genomic DNA
• B. Agarose gel electrophoresis of digested human genomic DNA
• C. Southern Blot transfer to nitrocellulose membrane
• D. Detect human AP homologues using a probe for human intestinal AP

Enrollment is limited .
Applications for admission may be picked up from Tynette, Room 6051, Medical School Building.