VOLUME 1
Preface ...................................................... xxix
Contributors ............................................... xxxiii
1. Escherichia coli, Plasmids, and Bacteriophages ............ 1-1
1.1. Medium Preparation and Bacteriological Tools ........ 1-2
Minimal Media ....................................... 1-2
Rich Media .......................................... 1-2
Solid Media ......................................... 1-3
Tools ............................................... 1-4
1.2. Growth in Liquid Media .............................. 1-5
Basic Protocol 1: Growing an Overnight Culture ...... 1-5
Basic Protocol 2: Growing Larger Cultures ........... 1-5
Basic Protocol 3: Monitoring Growth with a Count
Slide ............................................... 1-6
Basic Protocol 4: Monitoring Growth with a
Spectrophotometer ................................... 1-6
1.3. Growth on Solid Media ............................... 1-6
Basic Protocol 1: Titering and Isolating Bacterial
Colonies by Serial Dilutions ........................ 1-6
Basic Protocol 2: Isolating Single Colonies by
Streaking a Plate ................................... 1-7
Basic Protocol 3: Isolating Single Colonies by
Spreading a Plate ................................... 1-7
Basic Protocol 4: Replica Plating ................... 1-7
Support Protocol: Strain Storage and Revival ........ 1-8
1.4. Selected Topics from Classical Bacterial Genetics ... 1-8
1.5. Plasmid Vectors .................................... 1-14
Choosing a Plasmid Vector .......................... 1-14
1.6. Minipreps of Plasmid DNA ........................... 1-23
Basic Protocol 1: Alkaline Lysis Miniprep .......... 1-23
Alternate Protocol: Alkaline Lysis in 96-Well
Microtiter Dishes .................................. 1-24
Basic Protocol 2: Boiling Miniprep ................. 1-24
1.7. Large-Scale Preparation of Plasmid DNA ............. 1-25
Basic Protocol 1: Preparation of Crude Lysate by
Alkaline Lysis ..................................... 1-25
Basic Protocol 2: Purification of Plasmid DNA by
CsCl/Ethidium Bromide Equilibrium Centrifugation ... 1-26
Alternate Protocol: Purification of Plasmid DNA
by Anion-Exchange or Size-Exclusion
Chromatography ..................................... 1-27
1.8. Introduction of Plasmid DNA into Cells ............. 1-29
Basic Protocol 1: Transformation Using Calcium
Chloride ........................................... 1-29
Alternate Protocol: One-Step Preparation and
Transformation of Competent Cells .................. 1-30
Basic Protocol 2: High-Efficiency Transformation
by Electroporation ................................. 1-30
1.9. Introduction to Lambda Phages ...................... 1-32
Lytic Growth ....................................... 1-32
Lysogenic Growth ................................... 1-33
1.10. Lambda as a Cloning Vector ......................... 1-35
Advantages of Using Lambda ......................... 1-35
Selections for Inserted DNA ........................ 1-35
Maps of Lambda-Derived Cloning Vectors ............. 1-36
1.11. Plating Lambda Phage to Generate Plaques ........... 1-38
Basic Protocol 1: Isolating a Single Plaque by
Titering Serial Dilutions .......................... 1-38
Basic Protocol 2: Phage Transfection and In Vitro
Packaging .......................................... 1-39
1.12. Growing Lambda-Derived Vectors ..................... 1-40
Basic Protocol: Making a Phage Stock by Plate
Lysis .............................................. 1-40
Alternate Protocol: Making a Liquid Phage Lysate ... 1-41
1.13. Preparing Lambda DNA from Phage Lysates ............ 1-41
Basic Protocol: Preparing Lambda DNA by Step- and
Equilibrium-Gradient Centrifugation ................ 1-41
1.14. Introduction to Vectors Derived from Filamentous
Phages ............................................. 1-43
1.15. Preparing Single-Stranded DNA Using M13-Derived
Vectors ............................................ 1-46
Basic Protocol: Preparing Single-Stranded DNA
from Plasmids Using Helper Phage ................... 1-46
2. Preparation and Analysis of DNA ............................ 2-1
Introduction ........................................ 2-1
Electrophoresis and Its Applications ................ 2-1
Gels and Electric Circuits .......................... 2-2
2.1. Purification and Concentration of DNA from Aqueous
Solutions ........................................... 2-3
Basic Protocol: Phenol Extraction and Ethanol
Precipitation of DNA ................................ 2-3
Alternate Protocol 1: Precipitation of DNA Using
Isopropanol ......................................... 2-4
Support Protocol 1: Concentration of DNA Using
Butanol ............................................. 2-4
Support Protocol 2: Removal of Residual Phenol,
Chloroform, or Butanol by Ether Extraction .......... 2-4
Alternate Protocol 2: DNA Purification Using
Silica Membrane Spin Columns ........................ 2-5
Alternate Protocol 3: Purification and
Concentration of RNA and Dilute Solutions of DNA .... 2-5
Alternate Protocol 4: Removal of Low-Molecular-
Weight Oligonucleotides and Triphosphates by
Ethanol Precipitation ............................... 2-6
2.2. Purification of DNA by Anion-Exchange
Chromatography ...................................... 2-7
Basic Protocol ...................................... 2-7
2.3. Preparation of Genomic DNA from Mammalian Tissue .... 2-8
Basic Protocol ...................................... 2-8
2.4. Preparation of Genomic DNA from Plant Tissue ........ 2-9
Basic Protocol: Preparation of Plant DNA Using CsCl
Centrifugation ...................................... 2-9
Alternate Protocol: Preparation of Plant DNA Using
CTAB ............................................... 2-10
2.5. Preparation of Genomic DNA from Bacteria ........... 2-11
Basic Protocol: Miniprep of Bacterial Genomic
DNA ................................................ 2-11
Alternate Protocol: Large-Scale CsCl Preparation
of Bacterial Genomic DNA ........................... 2-12
Support Protocol: Removal of Polysaccharides from
Existing DNA Preparations .......................... 2-13
2.6. Agarose Gel Electrophoresis ........................ 2-13
Basic Protocol: Resolution of DNA Fragments on
Standard Agarose Gels .............................. 2-13
Support Protocol: Minigels and Midigels ............ 2-15
2.7. Pulsed-Field Gel Electrophoresis ................... 2-15
Basic Protocol: Field-Inversion Electrophoresis .... 2-15
Alternate Protocol: Chef Electrophoresis ........... 2-16
Support Protocol: Preparation of High-Molecular-
Weight DNA Samples and Size Markers ................ 2-17
2.8. Isolation and Purification of Large DNA
Restriction Fragments from Agarose Gels ............ 2-19
Basic Protocol 1: Electroelution from
Agarose Gels ....................................... 2-19
Basic Protocol 2: Electrophoresis onto NA-45
Paper .............................................. 2-20
Basic Protocol 3: Isolation of DNA Fragments
Using Low Gelling/Melting Temperature Agarose
Gels ............................................... 2-21
Alternate Protocol 1: Recovery of DNA from Low
Gelling/Melting Temperature Agarose Gels Using
λ-Agarase Digestion ................................ 2-21
Alternate Protocol 2: Recovery of DNA from
Agarose Using Silica Membrane Spin Columns ......... 2-22
Support Protocol: Rapid Estimation of DNA
Concentration by Ethidium Bromide Dot
Quantitation ....................................... 2-23
2.9. Separation of Small DNA Fragments by Conventional
Gel Electrophoresis ................................ 2-23
Basic Protocol 1: Nondenaturing Polyacrylamide
Gel Electrophoresis ................................ 2-23
Alternate Protocol: Electroelution of Small DNA
Fragments from Polyacrylamide Gels ................. 2-25
Basic Protocol 2: Sieving Agarose Gel
Electrophoresis .................................... 2-26
2.10. Capillary Electrophoresis of DNA ................... 2-26
Instrumentation .................................... 2-26
Separation Theory .................................. 2-27
Strategic Planning ................................. 2-28
Basic Protocol 1: Separation of Oligonucleotides ... 2-29
Basic Protocol 2: Quantitative PCR Analysis ........ 2-31
Alternate Protocol: Genotyping ..................... 2-32
2.11. Southern Blotting .................................. 2-32
Basic Protocol: Southern Blotting onto a Nylon or
Nitrocellulose Membrane with High-Salt Buffer ...... 2-33
Support Protocol: Calibration of a UV
Transilluminator ................................... 2-35
Alternate Protocol 1: Southern Blotting onto a
Nylon Membrane with an Alkaline Buffer ............. 2-35
Alternate Protocol 2: Southern Blotting by
Downward Capillary Transfer ........................ 2-36
Alternate Protocol 3: Electroblotting from a
Polyacrylamide Gel to a Nylon Membrane ............. 2-37
2.12. Dot and Slot Blotting of DNA ....................... 2-38
Basic Protocol: Dot and Slot Blotting of DNA onto
Uncharged Nylon and Nitrocellulose Membranes
Using a Manifold ................................... 2-38
Alternate Protocol 1: Dot and Slot Blotting of
DNA onto a Positively Charged Nylon Membrane
Using a Manifold ................................... 2-39
Alternate Protocol 2: Manual Preparation of a
DNA Dot Blot ....................................... 2-40
2.13. Hybridization Analysis of DNA Blots ................ 2-40
Basic Protocol: Hybridization Analysis of a DNA
Blot with a Radiolabeled DNA Probe ................. 2-42
Alternate Protocol: Hybridization Analysis of
a DNA Blot with a Radiolabeled RNA Probe ........... 2-43
Support Protocol: Removal of Probes from
Hybridized Membranes ............................... 2-44
2.14. Synthesis and Purification of Oligonucleotides ..... 2-48
Introduction to Chemical Nucleic Acid Synthesis .... 2-48
Strategies for Nucleic Acid Synthesis .............. 2-51
Strategies for Oligonucleotide Purification ........ 2-52
2.15. Purification of Oligonucleotides Using Denaturing
Polyacrylamide Gel Electrophoresis ................. 2-53
Basic Protocol ..................................... 2-53
3. Enzymatic Manipulation of DNA and RNA ..................... 3-1
3.1. Digestion of DNA with Restriction Endonucleases ..... 3-2
Basic Protocol: Digesting a Single DNA Sample
with a Single Restriction Endonuclease .............. 3-2
Alternate Protocol 1: Digesting DNA with Multiple
Restriction Endonucleases ........................... 3-4
Alternate Protocol 2: Digesting Multiple Samples
of DNA .............................................. 3-4
Alternate Protocol 3: Partial Digestion of DNA
with Restriction Endonucleases ...................... 3-6
Support Protocol: Methylation of DNA ................ 3-7
3.2. Reagents and Radioisotopes Used to Manipulate
Nucleic Acids ....................................... 3-8
Stock Solutions ..................................... 3-8
Enzyme Reaction Conditions and Applications ......... 3-8
lOx Enzyme Buffers .................................. 3-8
Nucleoside Triphosphates ........................... 3-13
Radioisotopes for Labeling Nucleic Acids ........... 3-14
Basic Protocol: Measuring Radioactivity in DNA
and RNA by Acid Precipitation ...................... 3-15
Alternate Protocol: Spin-Column Procedure for
Separating Radioactively Labeled DNA from
Unincorporated dNTPs ............................... 3-15
3.3. DNA-Dependent DNA Polymerases ...................... 3-17
Escherichia coli DNA Polymerase I .................. 3-17
Basic Protocol 1: Uniform Labeling of DNA by
Nick Translation ................................... 3-17
Klenow Fragment of Escherichia coli DNA
Polymerase I ....................................... 3-18
Basic Protocol 2: Labeling 3' Ends of DNA .......... 3-18
Basic Protocol 3: Repairing 3' or 5' Overhanging
Ends to Generate Blunt Ends ........................ 3-19
Basic Protocol 4: Labeling of DNA by Random
Oligonucleotide-Primed Synthesis ................... 3-19
T4 DNA Polymerase .................................. 3-20
Native T7 DNA Polymerase ........................... 3-21
Modified T7 DNA Polymerase ......................... 3-22
Taq DNA Polymerase ................................. 3-22
3.4. Template-Independent DNA Polymerases ............... 3-23
Terminal Deoxynucleotidyltransferase
(Terminal Transferase) ............................. 3-23
3.5. RNA-Dependent DNA Polymerases ...................... 3-24
Reverse Transcriptase .............................. 3-24
3.6. DNA-Dependent RNA Polymerases ...................... 3-24
Phage RNA Polymerases: Sp6, T7, T3 ................. 3-24
3.7. Phosphatases and Kinases ........................... 3-25
Bacterial Alkaline Phosphatase and Calf Intestine
Phosphatase ........................................ 3-25
T4 Polynucleotide Kinase ........................... 3-25
3.8. Exonucleases ....................................... 3-26
Exonuclease VII (exo VII) .......................... 3-26
Lambda Exonuclease (λ exo) ......................... 3-26
T7 Gene 6 Exonuclease .............................. 3-27
Exonuclease III (exo III) .......................... 3-27
3.9. Endonucleases ...................................... 3-27
Bal 31 Nuclease .................................... 3-27
S1 Nuclease ........................................ 3-28
Mung Bean Nuclease ................................. 3-29
Micrococcal Nuclease ............................... 3-29
Deoxyribonuclease I (DNase I) ...................... 3-29
3.10. Ribonucleases ...................................... 3-30
Ribonuclease A ..................................... 3-30
Ribonuclease H ..................................... 3-31
Ribonuclease T1 .................................... 3-31
3.11. DNA Ligases ........................................ 3-32
T4 DNA Ligase ...................................... 3-32
Escherichia coli DNA Ligase ........................ 3-32
3.12. RNA Ligases ........................................ 3-33
T4 RNA Ligase ...................................... 3-33
3.13. Subcloning of DNA Fragments ........................ 3-33
Basic Protocol: Subcloning of DNA Fragments ........ 3-33
Alternate Protocol: Ligation of DNA Fragments in
Gel Slices ......................................... 3-35
3.14. Constructing Recombinant DNA Molecules by the
Polymerase Chain Reaction .......................... 3-36
Basic Protocol: Subcloning DNA Fragments ........... 3-36
3.15. Labeling and Colorimetric Detection of
Nonisotopic Probes ................................. 3-40
Basic Protocol 1: Preparation of Biotinylated
Probes by Nick Translation ......................... 3-40
Basic Protocol 2: Preparation of Biotinylated
Probes by Random Oligonucleotide-Primed
Synthesis .......................................... 3-41
Support Protocol: Colorimetric Detection of
Biotinylated Probes ................................ 3-42
Alternate Protocol: Preparation and Detection of
Digoxigenin-Labeled DNA Probes ..................... 3-43
3.16. Chemiluminescent Detection of Nomsotopic Probes .... 3-43
Basic Protocol: Chemiluminescent Detection of
Biotinylated Probes ................................ 3-43
Alternate Protocol: Chemiluminescent Detection
of Digoxigenin-Labeled Probes ...................... 3-46
Support Protocol: Calibrating an Ultraviolet
Light Source ....................................... 3-46
4. Preparation and Analysis of RNA ........................... 4-1
4.1. Guanidinium Methods for Total RNA Preparation ....... 4-2
Basic Protocol: Single-Step RNA Isolation from
Cultured Cells or Tissues ........................... 4-2
Alternate Protocol 1: CsCl Purification of RNA
from Cultured Cells ................................. 4-3
Alternate Protocol 2: CsCl Purification of RNA
from Tissue ......................................... 4-4
4.2. Phenol/SDS Method for Plant RNA Preparation ......... 4-5
Basic Protocol ...................................... 4-5
4.3. Preparation of Bacterial RNA ........................ 4-6
Basic Protocol 1: Isolation of High-Quality RNA
from Gram-Negative Bacteria ......................... 4-6
Alternate Protocol: Rapid Isolation of RNA from
Gram-Negative Bacteria .............................. 4-8
Basic Protocol 2: Isolation of RNA from
Gram-Positive Bacteria .............................. 4-8
4.4. Preparation of Poly(A)+RNA .......................... 4-9
Basic Protocol ...................................... 4-9
4.5. S1 Analysis of Messenger RNA Using Single-Stranded
DNA Probes ......................................... 4-11
Basic Protocol: S1 Analysis of mRNA Using
M13 Template ....................................... 4-11
Alternate Protocol 1: Synthesis of Single-
Stranded Probe from Double-Stranded Plasmid
Template ........................................... 4-13
Alternate Protocol 2: Quantitative S1 Analysis
of mRNA Using Oligonucleotide Probes ............... 4-14
Support Protocol: Controls for Quantitative S1
Analysis of mRNA ................................... 4-15
4.6. Ribonuclease Protection Assay ...................... 4-15
Basic Protocol ..................................... 4-15
Support Protocol 1: Preparation of Template DNA .... 4-17
Support Protocol 2: Gel Purification of RNA
Probes ............................................. 4-17
4.7. Primer Extension ................................... 4-18
Basic Protocol ..................................... 4-18
4.8. Analysis of RNA by Northern and Slot Blot
Hybridization ...................................... 4-20
Basic Protocol: Northern Hybridization of RNA
Fractionated by Agarose-Formaldehyde Gel
Electrophoresis .................................... 4-20
Alternate Protocol 1: Northern Hybridization of
RNA Denatured by Glyoxal/DMSO Treatment ........... 4-22
Alternate Protocol 2: Northern Hybridization of
Unfractionated RNA Immobilized by Slot Blotting .... 4-23
Support Protocol: Removal of Probes from Northern
Blots .............................................. 4-24
4.9. Identification of Newly Transcribed RNA ............ 4-25
Basic Protocol: Nuclear Runoff Transcription in
Mammalian Cells .................................... 4-25
Alternate Protocol 1: Isolation of Nuclei by
Dounce Homogenization .............................. 4-27
Alternate Protocol 2: Isolation of Nuclei by
Sucrose Gradient Centrifugation .................... 4-28
Support Protocol: Preparation of Nitrocellulose
Filters for Nuclear Runoff Transcription Assay ..... 4-29
5. Construction of Recombinant DNA Libraries ................. 5-1
5.1. Overview of Genomic DNA Libraries ................... 5-3
Representation and Randomness ....................... 5-3
Subgenomic DNA Libraries ............................ 5-3
Vectors for Genomic DNA Libraries ................... 5-4
5.2. Overview of cDNA Libraries .......................... 5-4
Basic Protocol ...................................... 5-5
5.4. Amplification of Cosmid and Plasmid Libraries ....... 5-6
Basic Protocol ...................................... 5-6
6. Screening of Recombinant DNA Libraries .................... 6-1
6.1. Plating and Transferring Bacteriophage Libraries .... 6-3
Basic Protocol: Plating and Transferring
Bacteriophage Libraries ............................. 6-3
6.2. Plating and Transferring Cosmid and Plasmid
Libraries ........................................... 6-4
Basic Protocol: Plating and Transferring Cosmid
and Plasmid Libraries ............................... 6-4
6.3. Using DNA Fragments as Probes ....................... 6-6
Basic Protocol: Hybridization in Formamide .......... 6-6
Alternate Protocol: Hybridization in Aqueous
Solution ............................................ 6-7
6.4. Using Synthetic Oligonucleotides as Probes .......... 6-7
Basic Protocol: Hybridization in Sodium
Chloride/Sodium Citrate (SSC) ....................... 6-7
Alternate Protocol: Hybridization in
Tetramethylammonium Chloride (TMAC) ................. 6-8
Support Protocol: Labeling the 5' Ends of Mixed
Oligonucleotides ................................... 6-10
6.5. Purification of Bacteriophage Clones ............... 6-11
Basic Protocol ..................................... 6-11
6.6. Purification of Cosmid and Plasmid Clones .......... 6-12
Basic Protocol ..................................... 6-12
6.7. Immunoscreening of Fusion Proteins Produced in
Lambda Plaques ..................................... 6-12
Basic Protocol: Screening a λgt11 Expression
Library with Antibodies ............................ 6-12
Alternate Protocol: Induction of Fusion Protein
Expression with IPTG Prior to Screening with
Antibodies ......................................... 6-13
6.8. Immunoscreening After Hybrid Selection and
Translation ........................................ 6-14
Basic Protocol ..................................... 6-14
6.9. Use of Monoclonal Antibodies for Expression
Cloning ............................................ 6-16
Basic Protocol: Isolation of cDNA Clones Encoding
Cell-Surface Antigens .............................. 6-16
Support Protocol 1: Preparation of
Antibody-Coated Plates ............................. 6-19
Alternate Protocol: Isolation of cDNA Clones
Encoding Intracellular Antigens .................... 6-19
Support Protocol 2: Preparation of Poly
vinylidene-Wrapped Plates .......................... 6-21
6.10. Recombination-Based Assay (RBA) for Screening
Bacteriophage Lambda Libraries ..................... 6-22
Basic Protocol ..................................... 6-22
7. DNA Sequencing ............................................ 7-1
Introduction .............................................. 7-1
Dideoxy (Sanger) Sequencing ............................... 7-1
Chemical (Maxam-Gilbert) Sequencing ....................... 7-4
Choosing Between Dideoxy and Chemical Sequencing
Methods ................................................... 7-5
Alternatives to Radiolabeled Sequencing Reactions ......... 7-6
Other Developments in Sequencing Technology ............... 7-6
Computer Analysis ......................................... 7-7
7.1. DNA Sequencing Strategies ........................... 7-7
Dideoxy Sequencing .................................. 7-8
Chemical Sequencing ................................ 7-11
7.2. Constructing Nested Deletions for Use in DNA
Sequencing ......................................... 7-12
Basic Protocol 1: Using Exonuclease III to
Construct Unidirectional Deletions ................. 7-12
Alternate Protocol: Protection of DNA from
Exonuclease III Digestion Using [a-35S]dNTPs ....... 7-16
Basic Protocol 2: Using Bal 31 Nuclease to
Construct Nested Deletions ......................... 7-16
7.3. Preparation of Templates for DNA Sequencing ........ 7-20
Basic Protocol 1: Preparation of Single-Stranded
M13 Phage DNA ...................................... 7-20
Basic Protocol 2: Preparation of A, DNA from
Small-Scale Lysates ................................ 7-21
Basic Protocol 3: Miniprep of Recombinant pSP64CS
or pSP65CS Plasmid DNA for Chemical Sequencing ..... 7-22
Basic Protocol 4: Miniprep of Double-Stranded
Plasmid DNA for Dideoxy Sequencing ................. 7-23
Basic Protocol 5: Alkaline Denaturation of
Double-Stranded Plasmid or λ DNA for Dideoxy
Sequencing ......................................... 7-24
Basic Protocol 6: Preparation of Plasmid DNA
from an E. coli Colony or Phage DNA from a
Plaque for Thermal Cycle Sequencing ................ 7-24
7.4. DNA Sequencing by the Dideoxy Method ............... 7-25
Basic Protocol 1: Labeling/Termination
Reactions Using Sequenase (Modified T7
DNA Polymerase) .................................... 7-25
Alternate Protocol 1: Labeling/Termination
Sequencing Reactions Using Sequenase and Mn2+ ...... 7-29
Alternate Protocol 2: Sanger Sequencing Reactions
Using Taq DNA Polymerase ........................... 7-29
Alternate Protocol 3: One-Step Sequencing
Reactions Using 5'-End-Labeled Primers ............. 7-30
Basic Protocol 2: Thermal Cycle Sequencing
Reactions Using a-Labeled Nucleotides .............. 7-31
Alternate Protocol 4: Thermal Cycle Sequencing
Reactions Using 5'-End-Labeled Primers ............. 7-33
Alternate Protocol 5: Cycle Sequencing Using
Fluorescence Dye-Labeled Primers and Terminators ... 7-33
7.5. Dideoxy DNA Sequencing with Chemiluminescent
Detection .......................................... 7-35
Basic Protocol: DNA Sequencing Using Biotinylated
Primers with Chemiluminescent Detection ............ 7-36
Alternate Protocol 1: Two-Step (Indirect)
Detection Using Streptavidin and Biotinylated
Alkaline Phosphatase ............................... 7-39
Alternate Protocol 2: Sequencing with Hapten-
Labeled Primers and Detection with Antibody-
Alkaline Phosphatase Conjugates .................... 7-39
7.6. DNA Sequencing by the Chemical Method .............. 7-40
Basic Protocol: Chemical Sequencing Using
12P-Labeled DNA .................................... 7-40
Support Protocol: Tth1111 Digestion and
End Labeling ....................................... 7-42
7.7. Denaturing Gel Electrophoresis for Sequencing ...... 7-44
Basic Protocol: Pouring, Running, and Processing
Sequencing Gels .................................... 7-45
Alternate Protocol 1: Buffer-Gradient Sequencing
Gels ............................................... 7-48
Alternate Protocol 2: Electrolyte-Gradient
Sequencing Gels .................................... 7-48
Alternate Protocol 3: Formamide-Containing
Sequencing Gels .................................... 7-49
8. Mutagenesis of Cloned DNA ................................. 8-1
8.1. Mutagenesis with Degenerate Oligonucleotides:
Creating Numerous Mutations in a Small DNA
Sequence ............................................ 8-2
Basic Protocol ...................................... 8-2
8.2. Gene Synthesis: Assembly of Target Sequences Using
Mutually Priming Long Oligonucleotides .............. 8-5
Basic Protocol ...................................... 8-5
8.3. Random Mutagenesis by PCR ........................... 8-7
Basic Protocol: Mutagenesis of a DNA Sequence ....... 8-8
Alternate Protocol: Mutagenesis of a Library
of Sequences ........................................ 8-9
8.4. Linker-Scanning Mutagenesis of DNA ................. 8-10
Basic Protocol ..................................... 8-10
8.5. Directed Mutagenesis by PCR ........................ 8-13
Basic Protocol 1: Introduction of Restriction
Endonuclease Sites by PCR .......................... 8-13
Basic Protocol 2: Introduction of Point Mutations
by PCR ............................................. 8-16
Alternate Protocol: Introduction of a Point
Mutation by Sequential PCR Steps ................... 8-19
9. Introduction of DNA into Mammalian Cells .................. 9-1
Introduction .............................................. 9-1
Choice of Transfection Method ............................. 9-1
Optimization of Transfection .............................. 9-2
Viral Vectors ............................................. 9-3
Mammalian Cell Culture .................................... 9-4
9.1. Calcium Phosphate Transfection ...................... 9-6
Basic Protocol: Transfection Using Calcium
Phosphate-DNA Precipitate Formed in HEPES ........... 9-6
Support Protocol: Glycerol/DMSO Shock of Mammalian
Cells ............................................... 9-7
Alternate Protocol: High-Efficiency Transfection
Using Calcium Phosphate-DNA Precipitate Formed
in BES .............................................. 9-7
9.2. Transfection Using Deae-Dextran ..................... 9-9
Basic Protocol: General Procedure for DEAE-Dextran
Transfection ........................................ 9-9
Alternate Protocol: Sample Experiment: Testing
Enzyme Structure/Activity Relationships ............ 9-10
9.3. Transfection by Electroporation .................... 9-11
Basic Protocol: Electroporation into Mammalian
Cells .............................................. 9-11
Alternate Protocol: Electroporation into Plant
Protoplasts ........................................ 9-12
9.4. Transfection of Cultured Eukaryotic Cells Using
Cationic Lipid Reagents ............................ 9-13
Basic Protocol 1: Cationic Lipid-Mediated
Transfection of Adherent Mammalian Cells
with DNA ........................................... 9-14
Alternate Protocol: Enhanced Cationic Lipid-
Mediated Transfection of Adherent Mammalian
Cells with DNA ..................................... 9-15
Basic Protocol 2: Cationic Lipid-Mediated
Transfection of Suspension Cells with DNA .......... 9-16
Basic Protocol 3: Cationic Lipid-Mediated
Transfection of Adherent Cells with RNA ............ 9-16
Basic Protocol 4: Cationic Lipid-Mediated
Transfection of Adherent Sf9 and Sf21 Insect
Cells with Baculovirus DNA .......................... 9-17
Support Protocol: Fine Tuning or Optimizing
Conditions for Cationic Lipid Reagent
Transfections ...................................... 9-18
9.5. Selection of Transfected Mammalian Cells ........... 9-19
Strategic Planning ................................. 9-19
Basic Protocol 1: Stable Transfer of Genes into
Mammalian Cells .................................... 9-21
Basic Protocol 2: Selectable Markers for
Mammalian Cells .................................... 9-23
9.6. Overview of Genetic Reporter Systems ............... 9-26
Design of Reporter Vectors ......................... 9-28
In Vitro Reporter Assays ........................... 9-31
In Vivo Reporter Assays ............................ 9-34
9.7. Isotopic Assays for Reporter Gene Activity ......... 9-35
Basic Protocol 1: Chromatographic Assay for CAT
Activity ........................................... 9-35
Alternate Protocol 1: In Situ Lysis of Cells for
CAT Assay .......................................... 9-37
Alternate Protocol 2: Phase-Extraction Assay for
CAT Activity ....................................... 9-38
Basic Protocol 2: Radioimmunoassay for Human
Growth Hormone ..................................... 9-39
9.8. Nonisotopic Assays for Reporter Gene Activity ...... 9-40
Basic Protocol 1: Firefly Luciferase Reporter
Gene Assay ......................................... 9-40
Alternate Protocol: Luciferase Assay in
Freeze-Thaw-Lysed Cells ............................ 9-41
Basic Protocol 2: Chemiluminescent
(3-Galactosidase Reporter Gene Assay ............... 9-43
9.9. Use of the A. Victoria Green Fluorescent Protein
to Study Protein Dynamics In Vivo .................. 9-44
Utilization of GFP ................................. 9-44
Problems with GFP .................................. 9-45
Mutants of GFP ..................................... 9-46
Microscopy Setup ................................... 9-46
9.10. Overview of the Retrovirus Transduction System ..... 9-47
Retrovirus Life Cycle .............................. 9-48
Replication-Competent Vectors ...................... 9-48
Replication-Incompetent Vectors .................... 9-48
Packaging Lines and Virus Production ............... 9-49
Murine Retroviruses ................................ 9-51
Avian Retroviruses ................................. 9-57
Safety Issues ...................................... 9-58
9.11. Preparation of a Specific Retrovirus Producer
Cell Line .......................................... 9-58
Basic Protocol 1: Introduction of a Retrovirus
Vector into a Packaging Cell Line .................. 9-58
Basic Protocol 2: Determination of Viral Titer:
Identification of Producer Clones Making
High-Titer Virus ................................... 9-61
Alternate Protocol: Rapid Evaluation of Producer
Colonies ........................................... 9-62
Support Protocol: Xgal Staining of Cultured
Cells .............................................. 9-62
9.12. Transient Transfection Methods for Preparation of
High-Titer Retroviral Supernatants ................. 9-63
Basic Protocol 1: Transient Transfection of a
Retrovirus Vector into 293 Cells ................... 9-63
Support Protocol: Growth and Storage
293 Cells .......................................... 9-64
Basic Protocol 2: Infection of Adherent Cells
with Retroviral Supernatant ........................ 9-65
Alternate Protocol 1: Infection of Adherent
Cells by Spin Infection ............................ 9-66
Basic Protocol 3: Infection of Nonadherent Cells
by Retroviral Supernatant .......................... 9-66
Alternate Protocol 2: Infection of Nonadherent
Cells by Co-Cultivation ............................ 9-67
Alternate Protocol 3: Infection of Nonadherent
Cells by Spin Infection ............................ 9-68
9.13. Large-Scale Preparation and Concentration of
Retrovirus Stocks .................................. 9-69
Basic Protocol: Preparation of Virus Stock and
Concentration by Centrifugation .................... 9-69
Alternate Protocol 1: Concentration by PEG
Precipitation and Chromatography ................... 9-70
Alternate Protocol 2: Concentration Using
Molecular-Weight-Cutoff Filters .................... 9-70
9.14. Detection of Helper Virus in Retrovirus Stocks ..... 9-71
Basic Protocol: Detection of Helper Virus Through
Horizontal Spread of Drug Resistance ............... 9-71
Alternate Protocol 1: Proviral Rescue to Detect
Replication-Competent Retrovirus ................... 9-72
Alternate Protocol 2: Reverse Transcriptase Assay
to Detect Helper Virus ............................. 9-72
9.15. Retrovirus Infection of Cells In Vitro and
In Vivo ............................................ 9-73
Infection of Cells In Vitro ........................ 9-73
Infection of Rodents and Chicks In Vivo ............ 9-75
10. Analysis of Proteins ..................................... 10-1
Introduction ............................................. 10-1
Classification of Proteins ............................... 10-1
Protein Purification Flow Charts ......................... 10-1
10.1. Spectrophotometric and Colorimetric Determination
of Protein Concentration ........................... 10-9
Basic Protocol 1: Using A280 to Determine Protein
Concentration ...................................... 10-9
Basic Protocol 2: Using the Bradford Method
to Determine Protein Concentration ................. 10-9
10.2. Quantitative Amino Acid Analysis .................. 10-10
Sample Preparation ................................ 10-10
Calculation of the Average Composition ............ 10-11
10.3. One-Dimensional SDS Gel Electrophoresis
of Proteins ....................................... 10-12
Electricity and Electrophoresis ................... 10-12
Basic Protocol 1: Denaturing (SDS) Discontinuous
PAGE: Laemmli Method .............................. 10-14
Alternate Protocol 1: PAGE in Tris-Tricine
Buffer Systems .................................... 10-18
Alternate Protocol 2: Nonurea Peptide
Separations with Tris Buffers ..................... 10-20
Alternate Protocol 3: Continuous SDS-PAGE ......... 10-21
Alternate Protocol 4: PAGE with Ultrathin Gels .... 10-22
Support Protocol 1: Casting Multiple
Single-Concentration Gels ......................... 10-23
Alternate Protocol 5: Separation of Proteins on
Gradient Gels ..................................... 10-25
Support Protocol 2: Casting Multiple Gradient
Gels .............................................. 10-27
Basic Protocol 2: Electrophoresis in
Single-Concentration Minigels ..................... 10-29
Support Protocol 3: Preparing Multiple Gradient
Minigels .......................................... 10-31
10.4. Two-Dimensional Gel Electrophoresis Using
the O'Farrell System .............................. 10-32
Basic Protocol 1: First-Dimension
(Isoelectric-Focusing) Gels ....................... 10-32
Alternate Protocol 1: Isoelectric Focusing of
Very Basic and Very Acidic Proteins ............... 10-34
Basic Protocol 2: Second-Dimension Gels ........... 10-35
Alternate Protocol 2: Two-Dimensional Minigels .... 10-37
Support Protocol: Solubilization and Preparation
of Proteins in Tissue Samples ..................... 10-37
10.5. Staining Proteins in Gels ......................... 10-38
Basic Protocol 1: Coomassie Blue Staining ......... 10-38
Alternate Protocol 1: Rapid Coomassie
Blue Staining ..................................... 10-39
Basic Protocol 2: Silver Staining ................. 10-39
Alternate Protocol 2: Nonammoniacal Silver
Staining .......................................... 10-40
Alternate Protocol 3: Rapid Silver Staining ....... 10-41
Support Protocol 1: Gel Photography of
Coomassie- or Silver-Stained Gels ................. 10-42
Basic Protocol 3: Fluorescent Staining ............ 10-43
Alternate Protocol 4: Fluorescent Staining on
Nondenaturing Gels ................................ 10-44
Support Protocol 2: Gel Photography of
Fluorescently Stained Gels ........................ 10-44
10.6. Immunoblotting and Immunodetection ................ 10-45
Basic Protocol 1: Protein Blotting with Tank
Transfer Systems .................................. 10-45
Alternate Protocol 1: Protein Blotting with
Semidry Systems ................................... 10-47
Alternate Protocol 2: Blotting of Stained Gels .... 10-48
Support Protocol 1: Reversible Staining of
Transferred Proteins .............................. 10-49
Basic Protocol 2: Immunoprobing with Directly
Conjugated Secondary Antibody ..................... 10-49
Alternate Protocol 3: Immunoprobing with
Avidin-Biotin Coupling to Secondary Antibody ...... 10-50
Basic Protocol 3: Visualization with Chromogenic
Substrates ........................................ 10-51
Alternate Protocol 4: Visualization with
Luminescent Substrates ............................ 10-51
Support Protocol 2: Stripping and Reusing
Membranes ......................................... 10-53
10.7. Gel-Filtration Chromatography ..................... 10-53
Basic Protocol 1: Desalting (Group Separation) .... 10-53
Basic Protocol 2: Protein Fractionation ........... 10-59
Basic Protocol 3: Determination of
Molecular Size .................................... 10-62
Support Protocol: Column Calibration .............. 10-64
10.8. Ion-Exchange Chromatography ....................... 10-67
Strategic Planning ................................ 10-67
Basic Protocol 1: Batch Adsorption and
Step-Gradient Elution with Increasing
Salt Concentration ................................ 10-68
Basic Protocol 2: Column Chromatography with
Linear Gradient Elution ........................... 10-71
Support Protocol: Test Tube Pilot Experiment to
Determine Starting Conditions for Ion-Exchange
Chromatography .................................... 10-73
10.9. Immunoaffinity Chromatography ..................... 10-78
Basic Protocol: Isolation of Soluble or
Membrane-Bound Antigens ........................... 10-78
Alternate Protocol 1: Batch Purification of
Antigens .......................................... 10-80
Alternate Protocol 2: Low-pH Elution of
Antigens .......................................... 10-81
10.10.Metal-Chelate Affinity Chromatography ............. 10-81
Basic Protocol: Native MCAC for Purification of
Soluble Histidine-Tail Fusion Proteins ............ 10-81
Alternate Protocol 1: Denaturing MCAC for
Purification of Insoluble Histidine-Tail
Fusion Proteins ................................... 10-84
Alternate Protocol 2: Solid-Phase Renaturation
of MCAC-Purified Proteins ......................... 10-86
Support Protocol: NTA Resin Regeneration .......... 10-86
10.11.HPLC of Peptides and Proteins: Preparation and
System Set-Up ..................................... 10-87
Properties of Peptides and Proteins and Their
Implications for HPLC Method Development........... 10-87
Detection of Peptides and Proteins in HPLC ........ 10-87
Start-Up Procedures ............................... 10-87
Preparation of the Sample ......................... 10-89
Preparation of the Mobile Phase ................... 10-90
Setting Up the HPLC Instrument .................... 10-90
Priming Pumps and Low-Pressure Lines with
Eluents ........................................... 10-90
Preparation of the HPLC System .................... 10-91
Gradient Delay (Dwell Volume) of the
HPLC System ....................................... 10-91
Connecting the Column ............................. 10-92
Programming the HPLC Instrument ................... 10-93
Injecting the Sample .............................. 10-93
Testing the Functional HPLC System ................ 10-93
Logbooks .......................................... 10-93
10.12.HPLC of Peptides and Proteins: Standard
Operating Conditions .............................. 10-94
Standard Operating Conditions for HP-SEC .......... 10-95
Standard Operating Conditions for HP-NPC .......... 10-96
Standard Operating Conditions for HP-HIC .......... 10-98
Standard Operating Conditions for HP-IEX .......... 10-99
Standard Operating Conditions for HP-HILIC ....... 10-100
Standard Operating Conditions for HP-IMAC ........ 10-101
Standard Operating Conditions for HP-BAC ......... 10-103
Standard Operating Conditions for RP-HPLC ........ 10-103
Desalting of Peptide and Protein Mixtures
by RP-HPLC Techniques ............................ 10-108
10.13.Reversed-Phase Isolation of Peptides ............. 10-109
Basic Protocol 1: Reversed-Phase Peptide
Separation at the 5- to 500-pmol Level ........... 10-109
Basic Protocol 2: Reversed-Phase Peptide
Separation at <5 pmol ............................ 10-110
Support Protocol: Capillary HPLC System
Assembly ......................................... 10-111
10.14.Purification of Recombinant Proteins and Study
of Protein Interaction by Epitope Tagging ........ 10-113
Basic Protocol: Immunoprecipitation of
Epitope-Tagged Recombinant Proteins .............. 10-113
10.15.Immunoprecipitation .............................. 10-115
Basic Protocol 1: Immunoprecipitation Using
Cells in Suspension Lysed with a Nondenaturing
Detergent Solution ............................... 10-115
Alternate Protocol 1: Immunoprecipitation
Using Adherent Cells Lysed with a Nondenaturing
Detergent Solution ............................... 10-119
Alternate Protocol 2: Immunoprecipitation
Using Cells Lysed with Detergent Under
Denaturing Conditions ............................ 10-119
Alternate Protocol 3: Immunoprecipitation Using
Cells Lysed Without Detergent .................... 10-120
Alternate Protocol 4: Immunoprecipitation with
Antibody-Sepharose ............................... 10-120
Support Protocol: Preparation of
Antibody-Sepharose ............................... 10-122
Alternate Protocol 5: Immunoprecipitation of
Radiolabeled Antigen with Anti-Ig Serum .......... 10-123
Basic Protocol 2: Immunoprecipitation-
Recapture ........................................ 10-125
10.16.Synthesizing Proteins In Vitro by Transcription
and Translation of Cloned Genes .................. 10-126
Basic Protocol ................................... 10-126
10.17.Metabolic Labeling with Amino Acids .............. 10-128
Safety Precautions for Working with 35S-Labeled
Compounds ........................................ 10-128
Basic Protocol: Pulse-Labeling of Cells in
Suspension with [35S]Methionine .................. 10-128
Alternate Protocol 1: Pulse-Labeling of
Adherent Cells with [15S]Methionine .............. 10-130
Alternate Protocol 2: Pulse-Chase Labeling
of Cells with [15S]Methionine .................... 10-130
Alternate Protocol 3: Long-Term Labeling of
Cells with [35S]Methionine ....................... 10-131
Alternate Protocol 4: Metabolic Labeling with
Other Radiolabeled Amino Acids ................... 10-132
Support Protocol: TCA Precipitation to
Determine Label Incorporation .................... 10-133
10.18.Isolation of Proteins for Microsequence
Analysis ......................................... 10-134
Basic Protocol 1: Determination of Amino Acid
Sequence by SDS-PAGE and Transfer to PVDF
Membranes ........................................ 10-134
Support Protocol: Preparation of Protein
Samples for SDS-PAGE ............................. 10-136
Basic Protocol 2: Determination of Internal
Amino Acid Sequence from N-Terminally Blocked
Proteins ......................................... 10-136
10.19.Capillary Electrophoresis of Proteins and
Peptides ......................................... 10-138
Instrumentation .................................. 10-138
Strategic Planning ............................... 10-140
Basic Protocol 1: Separation of Proteins by
Isoelectric Focusing ............................. 10-140
Basic Protocol 2: Separation of Proteins ......... 10-141
Basic Protocol 3: Analytical Peptide
Separations ...................................... 10-142
Basic Protocol 4: Micropreparative Capillary
Electrophoresis: Multiple Separations ............ 10-144
Alternate Protocol: Micropreparative Capillary
Electrophoresis: Single Separation ............... 10-145
11. Immunology ........................................... 11-1
11.1. Conjugation of Enzymes to Antibodies ............... 11-3
Basic Protocol: Conjugation of Horseradish
Peroxidase to Antibodies ........................... 11-3
Alternate Protocol: Conjugation of Alkaline
Phosphatase to Antibodies .......................... 11-4
11.2. Enzyme-Linked Immunosorbent Assays (ELISA) ......... 11-5
Basic Protocol: Indirect ELISA to Detect Specific
Antibodies ......................................... 11-6
Alternate Protocol 1: Direct Competitive ELISA
to Detect Soluble Antigens ......................... 11-7
Alternate Protocol 2: Antibody-Sandwich ELISA
to Detect Soluble Antigens ......................... 11-8
Alternate Protocol 3: Double Antibody-Sandwich
ELISA to Detect Specific Antibodies ................ 11-9
Alternate Protocol 4: Direct Cellular ELISA to
Detect Cell-Surface Antigens ...................... 11-10
Alternate Protocol 5: Indirect Cellular ELISA to
Detect Antibodies Specific for Surface Antigens ... 11-11
Support Protocol 1: Criss-Cross Serial Dilution
Analysis to Determine Optimal Reagent
Concentrations .................................... 11-12
Support Protocol 2: Preparation of Bacterial
Cell Lysate Antigens .............................. 11-12
11.3. Immunization of Mice .............................. 11-13
Basic Protocol: Production of Immune Spleen
Cells: Immunization with Soluble Antigen .......... 11-13
Alternate Protocol 1: Immunization with Complex
Antigens (Membranes, Whole Cells, and
Microorganisms) ................................... 11-14
Alternate Protocol 2: Immunization with Antigen
Isolated by Electrophoresis ....................... 11-15
11.4. Production of Monoclonal Antibody Supernatant
and Ascites Fluid ................................. 11-15
Basic Protocol 1: Production of a Monoclonal
Antibody Supernatant .............................. 11-16
Alternate Protocol 1: Large-Scale Production of
Monoclonal Antibody Supernatant ................... 11-16
Alternate Protocol 2: Large-Scale Production of
Hybridomas or Cell Lines .......................... 11-17
Basic Protocol 2: Production of Ascites Fluid
Containing Monoclonal Antibody .................... 11-17
11.5. Purification of Monoclonal Antibodies ............. 11-19
Basic Protocol: Purification Using Protein
A-Sepharose ....................................... 11-19
Alternate Protocol 1: Alternative Buffer System
for Protein A-Sepharose ........................... 11-20
Alternate Protocol 2: Purification by
Antigen-Sepharose and Anti-Mouse-Ig-Sepharose ..... 11-20
11.6. Production of Polyclonal Antisera ................. 11-21
Basic Protocol: Immunization to Produce
Polyclonal Antibodies Using Freund's Adjuvant ..... 11-21
Alternate Protocol: Immunization to Produce
Polyclonal Antiserum Using Other Adjuvants ........ 11-23
Support Protocol: Preparation of Serum
from Blood ........................................ 11-24
11.7. Purification of Immunoglobulin G Fraction from
Antiserum, Ascites Fluid, or Hybridoma
Supernatant ....................................... 11-24
Basic Protocol: Precipitation of IgG with
Saturated Ammonium Sulfate ........................ 11-24
Alternate Protocol: Fractionation of IgG by
Chromatography on DEAE-Affi-Gel Blue .............. 11-25
11.8. Selection of an Immunogenic Peptide ............... 11-26
11.9. Production of Antipeptide Antibodies .............. 11-28
Basic Protocol: Chemical Coupling of Synthetic
Peptide to Carrier Protein Using MBS .............. 11-28
Alternate Protocol: Chemical Coupling of
Synthetic Peptide to Carrier Protein Using
Glutaraldehyde .................................... 11-29
12. DNA-Protein Interactions ................................. 12-1
12.1. Preparation of Nuclear and Cytoplasmic Extracts
from Mammalian Cells ............................... 12-3
Basic Protocol: Preparing Nuclear Extracts ......... 12-3
Support Protocol 1: Optimization of Nuclear
Extraction ......................................... 12-6
Support Protocol 2: Preparation of the
Cytoplasmic (S-100) Fraction ....................... 12-7
12.2. Mobility Shift DNA-Binding Assay Using Gel
Electrophoresis .................................... 12-7
Basic Protocol: Mobility Shift Assay ............... 12-8
Alternate Protocol 1: Competition Mobility Shift
Assay ............................................. 12-11
Alternate Protocol 2: Antibody Supershift
Assay ............................................. 12-11
Alternate Protocol 3: Multicomponent Mobility
Shift Assays ...................................... 12-11
12.3. Methylation and Uracil Interference Assays for
Analysis of Protein-DNA Interactions .............. 12-13
Basic Protocol 1: Methylation Interference
Assay ............................................. 12-13
Basic Protocol 2: Uracil Interference Assay ....... 12-15
12.4. DNase I Footprint Analysis of Protein-DNA
Binding ........................................... 12-17
Basic Protocol: DNase I Footprint Titration ....... 12-17
Support Protocol: Quantitation of Protein-
Binding Equilibria by Densitometric and
Numerical Analyses ................................ 12-20
Alternate Protocol: DNase Footprinting in Crude
Fractions ......................................... 12-23
12.5. UV Crosslinking of Proteins to Nucleic Acids ...... 12-24
Basic Protocol: UV Crosslinking Using a
Bromodeoxyuridine-Substituted Probe ............... 12-24
Alternate Protocol 1: UV Crosslinking Using a
Non-Bromodeoxyuridine-Substituted Probe ........... 12-26
Alternate Protocol 2: UV Crosslinking In Situ ..... 12-27
12.6. Purification of DNA-Binding Proteins Using
Biotin/Streptavidin Affinity Systems .............. 12-27
Basic Protocol: Purification of DNA-Binding
Proteins Using Biotin/Streptavidin Affinity
Systems ........................................... 12-27
Alternate Protocol 1: Purification Using a
Microcolumn ....................................... 12-30
Alternate Protocol 2: Purification Using
Streptavidin-Agarose .............................. 12-30
12.7. Detection, Purification, and Characterization
of cDNA Clones Encoding DNA-Binding Proteins ...... 12-31
Basic Protocol: Screening a λgt11 Expression
Library with Recognition-Site DNA ................. 12-32
Alternate Protocol: Denaturation/Renaturation
Cycling of Dried Replica Filters Using
Guanidine⋅HCl ..................................... 12-34
Support Protocol: Preparation of a Crude
Extract from Recombinant Lysogen to
Characterize DNA-Binding Activity ................. 12-35
12.8. Analysis of DNA-Protein Interactions Using
Proteins Synthesized In Vitro from Cloned Genes ... 12-36
Basic Protocol .................................... 12-36
12.9. Purification of Sequence-Specific DNA-Binding
Proteins by Affinity Chromatography ............... 12-37
Basic Protocol 1: Preparation of DNA
Affinity Resin .................................... 12-37
Alternate Protocol: Coupling the DNA to
Commercially Available CNBr-Activated Sepharose ... 12-40
Support Protocol 1: Purification of
Oligonucleotides by Preparative Gel
Electrophoresis ................................... 12-41
Basic Protocol 2: DNA Affinity Chromatography ..... 12-42
Support Protocol 2: Selection and Preparation
of Nonspecific Competitor DNA ..................... 12-45
12.10.Determination of Protein-DNA Sequence Specificity
by PCR-Assisted Binding Site Selection ............ 12-45
Basic Protocol .................................... 12-45
Support Protocol: Isolation and Analysis
of Bound Oligonucleotides from Mobility
Shift Gels ........................................ 12-50
Appendices
A1 Reagents and Solutions ............................. Al-1
References ............................................ 1
Index
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