Protocols and Applications Guide (US Letter Size) - Promega

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CONTENTS
I. Introduction 1
A. Basic Isolation Procedure 1
B. Basis for Purification by Silica 1
C. Overview of Plasmid DNA Purification 2
D. Overview of Genomic DNA Isolation 3
E. Overview of DNA Fragment Purification from Agarose
Gels and PCR Amplifications 3
F. Overview of Personal Automation Systems for
Purification 4
G. Methods for Determining DNA Yield and Purity 4
H. Estimation of DNA Concentration, Yield and Purity by
Absorbance 6
II. General Considerations for Plasmid DNA
Purification 6
A. Bacterial Growth and Culture Conditions 6
B. Antibiotic Selection 7
C. Recommended Inoculation Procedures 7
III. Factors That Affect Plasmid DNA Quality and
Yield 8
A. Bacterial Strain Selection 8
B. Plasmid Copy Number 8
C. Appropriate Sample Size and Throughput 9
D. Biomass Processed 9
E. Plasmid Purification Method and Transfection 9
IV. Plasmid DNA Purification Systems 10
A. PureYield Plasmid Miniprep System 10
B. PureYield Plasmid Midiprep System 12
C. PureYield Plasmid Maxiprep System 13
D. Wizard® Plus SV Minipreps DNA Purification
System 13
E. Wizard® SV 96 and SV 9600 Plasmid DNA Purification
Systems 14
F. Wizard® MagneSil® Plasmid Purification System 15
G. Wizard MagneSil Tfx System 15
V. Plasmid DNA Purification Protocol Featuring the
PureYield Plasmid Midiprep System 16
A. Standard DNA Purification Protocol 16
VI. Genomic DNA Isolation Systems 18
A. Wizard® Genomic DNA Purification Kit 18
B. Wizard® SV Genomic DNA Purification System 18
C. Wizard® SV 96 Genomic DNA Purification System 20
D. MagneSil® Genomic Systems for Blood Isolation 21
Protocols & Applications Guide
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E. Maxwell® 16 System 22
F. Plant Genomic DNA Isolation 24
G. Food DNA Isolation 25
H. Fixed-Tissue Genomic DNA Isolation 25
VII. Genomic DNA Purification Protocols Featuring the
Wizard® Genomic DNA Purification Kit 27
A. Isolation of Genomic DNA from Whole Blood 27
B. Isolation of Genomic DNA from Tissue Culture Cells and
Animal Tissue 28
C. Isolation of Genomic DNA from Gram-Positive and
Gram-Negative Bacteria 28
D. Isolation of Genomic DNA from Yeast Cultures or Plant
Tissue 29
VIII.Specialized Genomic DNA Purification Protocol
Featuring the MagneSil® Genomic, Large Volume
System 29
A. Sample Lysis and DNA Binding 30
B. Sample Washing 30
C. Elution of Purified Genomic DNA 31
IX. Fragment/PCR Product Purification Systems 31
A. Wizard® SV Gel and PCR Clean-Up System 31
B. Wizard® SV 96 PCR Clean-Up System 32
C. BigDye® Sequencing Clean-Up 33
X. Fragment/PCR Product Purification Protocol
Featuring the Wizard® SV Gel and PCR Clean-Up
System 34
A. Preparing the Membrane Wash Solution 34
B. DNA Purification by Centrifugation 34
C. DNA Purification by Vacuum 34
XI. Composition of Solutions 35
XII. References 35
PROTOCOLS & APPLICATIONS GUIDE