Syllabus

Recombinant DNA Technology (BIO 680/560)

(Mon. & Thurs., 5:00 PM to 8:30 PM)

Textbook/Lab Manual/Lab Notebook (all-in-one): Unraveling DNA: Molecular Biology for the Laboratory. Michael R. Winfrey, Marc A. Rott & Alan T. Wortman. Prentice Hall, Upper Saddle River, NJ, 1997. The schedule below is "keyed" to the exercise numbers in this textbook.
The design of this course takes advantage of a few serendipitous properties of the Vibrio fischeri MJ1 genome. For example, the lux operon, encoding all of the enzymatic activities responsible for its bioluminescent phenotype, is contained within a 9 kb Sal I fragment. These properties greatly facilitate the "shotgun" cloning and characterization of the lux operon, as well as subcloning of luxA (actually luxAB) encoding the heterodimeric luciferase enzyme.

Date: Lab #: Ex. #: Title:

Jan. 3 1 1-4 Introduction, Review and Overview

Jan. 6 2 5 Isolation of Chromosomal DNA from Vibrio fischeri.

Jan. 10 3 6 Large-scale Purification of Plasmid DNA.

Jan. 13 4 7 Spectrophotometric Analysis of DNA.

8 Restriction Digestion of Vibrio fischeri Genomic DNA and Plasmid Vector.

Jan. 17 - - HOLIDAY

Jan. 20 5 9 Quantification of Genomic DNA by Fluorometry and Agarose Plate Fluorescence.

10 Ligation of Restriction Fragments of Vibrio fischeri DNA to a Plasmid Vector.

Jan. 24 6 12 Transformation of Competent Escherichia coli DH5a with Recombinant Plasmids.

Jan. 27 7 13 Screening the Vibrio fischeri Genomic Library for Light Producing Clones.

Jan. 31 8 14 Small-scale Plasmid Isolations (Mini-Preps) from Bioluminescent Clones.

Feb. 3 9 15 Restriction Mapping of Plasmids from Bioluminescent Clones.

Mid-Term Exam.

Feb. 7 10 16 Southern Blotting and Hybridization to Detect the luxA Gene.

Feb. 10 11 17 Restriction Digestion of lux Plasmids and Cloning Vector for Subcloning luxA

Feb. 14 12 18 Gel Purification of DNA Restriction Fragments Containing luxA.

Feb. 17 13 19 Subcloning luxA into a Plasmid Vector.

Feb. 21 - - HOLIDAY

Feb. 24 14 20 Transformation of Competent Escherichia coli DH5a with subcloned DNA.

Feb. 28 15 21 Colony Hybridization to Screen for luxA Subclones.

Mar. 2 16 23 Amplification of luxA from Natural Isolates by the Polymerase Chain Reaction (PCR).

Mar. 6 17 24 Southern Blotting and Hybridization of PCR Products.

Mar. 9 18 26 Computer Analysis of DNA Sequences Using the World Wide Web.

Final Exam

Date:	Lab #:	Ex. #:	Title:
Jan. 3	1	1-4	Introduction, Review and Overview
Jan. 6	2	5	Isolation of Chromosomal DNA from Vibrio fischeri.
Jan. 10	3	6	Large-scale Purification of Plasmid DNA.
Jan. 13	4	7	Spectrophotometric Analysis of DNA.
		8	Restriction Digestion of Vibrio fischeri Genomic DNA and Plasmid Vector.
Jan. 17	-	-	HOLIDAY
Jan. 20	5	9	Quantification of Genomic DNA by Fluorometry and Agarose Plate Fluorescence.
		10	Ligation of Restriction Fragments of Vibrio fischeri DNA to a Plasmid Vector.
Jan. 24	6	12	Transformation of Competent Escherichia coli DH5a with Recombinant Plasmids.
Jan. 27	7	13	Screening the Vibrio fischeri Genomic Library for Light Producing Clones.
Jan. 31	8	14	Small-scale Plasmid Isolations (Mini-Preps) from Bioluminescent Clones.
Feb. 3	9	15	Restriction Mapping of Plasmids from Bioluminescent Clones.
			Mid-Term Exam.
Feb. 7	10	16	Southern Blotting and Hybridization to Detect the luxA Gene.
Feb. 10	11	17	Restriction Digestion of lux Plasmids and Cloning Vector for Subcloning luxA
Feb. 14	12	18	Gel Purification of DNA Restriction Fragments Containing luxA.
Feb. 17	13	19	Subcloning luxA into a Plasmid Vector.
Feb. 21	-	-	HOLIDAY
Feb. 24	14	20	Transformation of Competent Escherichia coli DH5a with subcloned DNA.
Feb. 28	15	21	Colony Hybridization to Screen for luxA Subclones.
Mar. 2	16	23	Amplification of luxA from Natural Isolates by the Polymerase Chain Reaction (PCR).
Mar. 6	17	24	Southern Blotting and Hybridization of PCR Products.
Mar. 9	18	26	Computer Analysis of DNA Sequences Using the World Wide Web.
			Final Exam