Localization of Gene Expression Patterns in Transgenic Plants &
Identification of Homozygous DNA-Insertion Mutants in Arabidopsis

Experimental Molecular Biology SPRING 2010

You must come to lab prepared--this requires you to read over and think about the experimental protocols, not just print a copy of them. The procedures for each day are available here and you will be given any additional information in the pre-lab lectures.

SPECIAL NOTE: Record enough procedure details in your notebook during each day of lab so that you can repeat these procedures using your notebook as the ONLY resource (i.e., you should not use printed web pages or handouts from previous labs).

Catalogues from biological suppliers represent an accessible and free source of valuable information on reagents, equipment, and technical procedures. We order primarily from these companies:

General Reference:
Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

Day 1 PCR amplification of upstream regulatory sequence: Tues., 9 March

Lecture Topics		Experimental Procedures
Course introduction		Leaf DNA prep
Overview of research project		PCR of 5' regulatory region
		Pouring agarose gel

Day 2 Gel analysis and restriction digest of PCR product: Thurs., 11 March

Lecture Topics		Experimental Procedures
Agarose gel electrophoresis		Gel analysis of PCR product
Restriction enzymes		Clean-up of PCR product
		Restriction digest of PCR product

Day 3 DNA ligation and bacterial transformation: Tues., 16 March

Lecture Topics		Experimental Procedures
DNA ligation		Analytical gel of digested PCR product
Bacterial transformation		Ligation with GUS vector
		Transformation

Day 4 DNA mini preps and restriction enzyme analysis: Thurs., 18 March

Lecture Topics		Experimental Procedures
DNA mini preps		Plasmid mini preps
		Restriction digest of plasmid DNA
		Agarose gel analysis of digests

Day 5 GUS staining of transgenic Arabidopsis: Tues., 23 March

Lecture Topics		Experimental Procedures
Reporter genes		Staining of transgenic plants
GUS staining		Poster critique
Preparing a Scientific Poster

Day 6 Observation of GUS-stained plants: Thurs., 25 March (meet in BL 141, Dr. Caprette's laboratory)

Lecture Topics		Experimental Procedures
Preparing slides		Observation of plants under dissecting scope

Days 7 & 8 PCR screen of T-DNA mutants: Tues., 30 March & Tues., 6 April

Lecture Topics		Experimental Procedures
PCR mutant screen		Leaf DNA prep
		Primary PCR mutant screen
		Agarose gel

*Posters (printed and pdf) are due by 4 p.m. on Thursday, 15 April (in Biology 211C)
**Poster Q/A session is Thurs., 22 April (in Biology Basement B03)

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 21 July 2003
Updated 22 April 2009

Localization of Gene Expression Patterns in Transgenic Plants & Identification of Homozygous DNA-Insertion Mutants in Arabidopsis