cloning - Basics of Molecular Cloning: Instructors Manual...

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Unformatted text preview: Basics of Molecular Cloning: Instructors Manual 1. Purpose and Concepts Covered .........................................................................1 2. Protocol for Cloning the Luciferase Gene into the pGEM Vector ...................2 A. Excising the Luciferase Gene from Surrounding DNA.....................................2 B. Purifying the Gene Fragment...........................................................................4 C. Ligating the Gene into the Vector ....................................................................6 D. Transforming Bacteria with Your Clone ............................................................7 E. Expressing Luciferase in E. coli .......................................................................9 3. Composition of Buffers and Solutions .............................................................11 1. Purpose and Concepts Covered This introductory cloning laboratory is for use in courses that cover basic topics in molec- ular biology and genetics. Molecular cloning is a basic technique used in a molecular biology labs. In this manual, we include a protocol for isolating the luciferase gene from DNA using restriction digestion and cloning it into the multiple cloning region of a vector. The cloned luciferase gene is then expressed in E. coli . Promega Corporation 2800 Woods Hollow Road Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 Telephone 608-274-4330 Fax 608-277-2516 www.promega.com Printed in USA. IM006 3/10 Page 1 This instructors manual is avaliable online only. This teaching resource is made available free of charge by Promega Corporation. Reproduction permitted for noncommer- cial educational purposes only. Copyright 2010 Promega Corporation. All rights reserved. ! Page 2 Promega Corporation 2800 Woods Hollow Road Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 Telephone 608-274-4330 Fax 608-277-2516 www.promega.com IM006 Printed in USA. 3/10 2. Protocol for Cloning the Luciferase Gene into the pGEM Vector Preparation for the Laboratory Materials Required pGEM - luc DNA (Cat.# E1541) pGEM -4Z Vector (Cat.# P2161) BamHI restriction enzyme (Cat.# R6021; 10u/l) SacI restriction enzyme (Cat.# R6061; 10u/l) Buffer E (supplied with BamHI enzyme) Acetylated BSA (supplied with restriction enzymes) Agarose, LMP, Preparative Grade for Large Fragments (>1,000bp; Cat.# V2831) TBE Buffer, 10X, Molecular Biology Grade (Cat.# V4251; or have students make their own buffer) Ethidium Bromide Solution, Molecular Biology Grade (Cat.# H5041) Note: The gel fragment cleanup system will not work with DNA that has been stained with methylene blue....
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cloning - Basics of Molecular Cloning: Instructors Manual...

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