DIG Application Manual
for Filter Hybridization
last update March 17, 2001
Below is the table of contents for our DIG User's Manual . To select a chapter section, click on the corresponding page
number. The chapters below are in Acrobat PDF format and can be viewed, searched, and printed with the free Adobe
Acrobat Reader
Introduction
Introduction to the DIG System and the
DIG Application Manual
2
Chapter 1 · DIG Basics
1.
DIG Basics
Introduction
10
2.
DIG Basics
DIG Labeling
11
3.
DIG Basics
Evaluation of Probe Labeling Efficiency
16
4.
DIG Basics
Electrophoresis of Target Nucleic Acids on
Agarose Gels
19
5.
DIG Basics
Blot Transfer of Target Nucleic Acids to a
Membrane
21
6.
DIG Basics
Hybridization of Probe to Target
23
7.
DIG Basics
Stringent Washes of Blot
28
8.
DIG Basics
Detection of Probe-Target Hybrids
29
9.
DIG Basics
Stripping and Reprobing the Membrane
31
10.
DIG Basics
Equipping a DIG Labeling and Detection
Lab
33
11.
DIG Basics
DIG Product Selection Guide: The Choice is
Yours
36
Chapter 2 · Procedures for Nonradioactive Labeling and Detection
1.
Procedures for
Nonradioactive
Labeling and
Detection
Introduction
40
2.
Procedures for
Nonradioactive
Labeling and
Detection
Techniques for DIG Labeling of
Hybridization Probes
41
2.1
Random Primed Labeling of DNA Probes
(High Yield Method)
43
2.2
PCR Labeling of DNA Probes
51
2.3
Transcriptional Labeling of RNA Probes
62
2.4
DIG Oligonucleotide Labeling
73
2.5
Direct Addition of DIG to RNA or DNA
74
2.6
Estimation of Probe Yield by the Direct
Detection Procedure
79
3.
Procedures for
Nonradioactive
Labeling and
Detection
Techniques for Hybridization of
DIG-labeled Probes to a Blot
88
4.
Procedures for
Nonradioactive
Labeling and
Detection
Techniques for Detection of Hybridization
Probes on a Blot
110
5.
Procedures for
Nonradioactive
Labeling and
Detection
Techniques for Stripping and Reprobing a
Membrane
127
6.
Procedures for
Nonradioactive
Labeling and
Detection
High Volume Screening Applications for
DIG-labeled Proteins
130
Chapter 3 · Detection and Quantification with the Lumi-Imager
1.
Detection and
Quantification with
the Lumi-Imager
Introduction to the Lumi-Imager
150
Chapter 4 · Other Nonradioactive Assays
1.
Other
Nonradioactive
Assays
Introduction
160
2.
Other
Nonradioactive
Assays
Nonradioactive Western Blot Assay
161
3.
Other
Nonradioactive
Assays
Nonradioactive Telomere Length Assay
164
4.
Other
Nonradioactive
Assays
Nonradioactive RNase Protection Assay
168
5.
Other
Nonradioactive
Assays
Nonradioactive Gel Mobility Shift Assay
171
6.
Other
Nonradioactive
Assays
Direct Detection of a DIG-labeled DNA
173
Appendices
Appendix A
Summary of DIG Basics
1. DIG Labeling
178
2. Electrophoresis and Blot Transfer
179
3. Hybridization of Probe Target
180
4. Stringent Washes of Blot
182
5. Sensitivity of Alkaline Phosphatase
Substrates
183
Appendix B
Troubleshooting the DIG System
1. Possible Problems and Recommendation
184
2. How to Optimize Single Copy Gene Detection
Easily
193
Appendix C
Suggestions for Improving DIG Labeling
and Detection Results
196
Appendix D
Preparation of Solutions
205
Appendix E
Ordering Information, DIG System
Products
210