Protocols and Applications Guide (US Letter Size) - Promega

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CONTENTS
I. Introduction 1
II. Promega Products for Cloning 1
A. Thermostable DNA Polymerases 1
B. T-Cloning Vectors 3
C. Flexi® Vector Systems 6
D. Modifying and Restriction Enzymes 9
E. Competent Cells 11
III. PCR Cloning Protocols 12
A. Amplification, Analysis and PCR Cleanup 12
B. Ligation and Transformation 13
C. Screening of Transformants 14
D. Subcloning 16
IV. Cloning Protocol for the Flexi® Vector Systems 16
A. PCR Primer Design, Amplification and Cleanup 16
B. Restriction Digest of PCR Product and Acceptor Flexi®
Vector 16
C. Ligation of PCR Product and Acceptor Flexi® Vector 17
D. Screening Clones with SgfI and PmeI 17
E. Transfer of Protein-Coding Region 17
V. Supplemental Cloning Techniques 20
A. Converting a 5´ Overhang to a Blunt End 20
B. Converting a 3´ Overhang to a Blunt End 21
C. Dephosphorylation of 5´ Ends 21
VI. Composition of Solutions 22
VII. References 23
Protocols & Applications Guide
www.promega.com
rev. 3/09
PROTOCOLS & APPLICATIONS GUIDE