Cover
Molecular Biology Techniques: A Classroom Laboratory Manual
Copyright
About the Authors
Preface
Acknowledgments
Note to Instructors
Nomenclature
Introduction
Conceptual Outline for Experiments
Experimental Procedures
Part I: Manipulation of DNA
Part II: Screening Transformants
Part III: Expression, Detection, and Purification of Recombinant Proteins From Bacteria
Part IV: Analysis of mRNA Levels
Part V: Modulation of Gene Expression
General Operating Procedures
Laboratory Safety
General Helpful Tips While Working in the Lab
Part I:
Manipulation of DNA
Reference
Advanced Alternatives Within Part I
Advanced Alternatives, Part I—Manipulation of DNA
AA1.1 Clone egfp With Different Primers
Additional materials needed
Experimental flow
AA1.2 Clone egfp Without PCR
Additional materials needed
Experimental flow
AA1.3 Clone a Gene Other Than egfp
Additional materials needed
Experimental flow
AA1.4 Semiquantitative Agarose Gel Electrophoresis
Additional materials needed
Lab Session 1 Getting Oriented; Practicing With Micropipettes
I Equipment Checklist
II Micropipetting
III Laboratory Exercises
A Micropipetting Self-Test
Self-Test 1
Self-Test 2
Self-Test 3
Self-Test 4
B Preparing Bovine Serum Albumin Dilutions
C Performing a Nitrocellulose Spot Test
Discussion Questions
Lab Session 2 Purification and Digestion of Plasmid (Vector) DNA
I Introduction
A Alkaline Lysis
B Silica Adsorption
C DNA Quantification
II Introduction to Expression Vectors
A Principles of Gene Expression
B Expression Vectors
C Orientation and Reading Frame
D Orientation
E Reading Frame
III Laboratory Exercises
A Alkaline Lysis and Silica Adsorption Protocol
B DNA Quantification
Option 1: Using the Nanodrop
Option 2: Using a Standard Spectrophotometer
C Restriction Digestion of Expression Vector DNA pET-41a, a GST Fusion Protein Vector
Restriction Enzyme Digestions
Setting Up Digestion of Your pET-41a Vector With NcoI and NotI
Reference
Discussion Questions
Lab Session 3 Completion of Vector Preparation and Polymerase Chain Reaction Amplification of egfp
I Introduction
What is the Polymerase Chain Reaction?
Why Clone by Polymerase Chain Reaction?
TA Cloning
PCR Cloning by Incorporation of Restriction Sites
Cloning Synthetic Genes
II Laboratory Exercises
A Clean-Up of Digested pET-41a Vector
B Agarose Gel Electrophoresis
C Polymerase Chain Reaction Amplification of egfp
PCR Protocol
References
Discussion Questions
Lab Session 4 Preparation of Insert DNA (egfp) PCR Product
I Introduction
II Laboratory Exercises
A Spin Column Clean-up of PCR Product
B Quantification of Purified egfp PCR Product
C Restriction Digestion of egfp PCR Product
D Removing Enzymes and Cleaning Digested DNA Using a Spin Column
E Check PCR Reaction and Purification on an Agarose Gel
Protocol (Review Lab Session 3b)
F Calculations for Next Week
Discussion Questions
Lab Session 5 DNA Ligation and Transformation of Escherichia coli
I Introduction
A Ligation
B Transformation
II Laboratory Exercises
A Ligations and Ligation Controls
B Divalent Cation-Mediated Transformation
C Electrophoresis of Ligation Reactions
Reference
Discussion Questions
Part II:
Screening Transformants
Advanced Alternatives Within Part II
Advanced Alternatives, Part II—Screening Transformants
AA2.1 Omit the PCR Screen
AA2.2 Omit the Restriction Analysis
AA2.3 Choose Different Restriction Enzymes for Analysis
Additional materials
AA2.4 Design Your Own Primers for the PCR Screen
Additional materials
Lab Session 6 Screening of Transformants, Part I
Lab Session 6a Interim Laboratory Session
WARNING!!! DUMMY ENTRY
I Introduction
II Laboratory Exercises
A Counting Transformants and Transformation Efficiency
B Replica Plating and Inoculation of Overnight Cultures
Lab Session 6b Isolation of Miniprep DNA From Potential Transformants
I Introduction
II Laboratory Exercise
Lab Session 6c PCR Screening
I Introduction
II Laboratory Exercise
Lab Session 6d Visualization of Green Fluorescent Protein, Part I
I Introduction
II Laboratory Exercise
Discussion Questions
Lab Session 7 Screening of Transformants, Part II
Lab Session 7a Restriction Digestion of DNA
I Introduction
II Laboratory Exercise
Lab Session 7b Analysis of PCR Screen and Miniprep Digestions
I Introduction
II Laboratory Exercise
Lab Session 7c Visualization of Enhanced Green Fluorescent Protein, Part II
I Introduction
II Laboratory Exercise
Lab Session 7d Analysis of DNA Sequence from a Positive Clone, Part I
I Introduction
II Laboratory Exercise
III Compiled Screening Data
References
Discussion Questions
Lab Session 8 Analysis of DNA Sequence From a Positive Clone, Part II
I Introduction
II Laboratory Exercise
Reference
Discussion Questions
Part III:
Expression, Detection, and Purification of Recombinant Proteins From Bacteria
Advanced Alternatives Within Part III
Advanced Alternatives, Part III—Expression, Detection and Purification of Recombinant Proteins from Bacteria
AA3.1 Omit Performing a Western Blot on Whole Cell Lysates and Perform a Western Blot on Purification Fractions
AA3.2 Semi-quantitative Western Blot
Additional materials
AA3.3 Cleaving the GST Fusion Tag
Additional materials
Lab Session 9 Expression of Fusion Protein from Positive Clones, SDS–PAGE and Western Blot: Part I
Lab Session 9a Interim Laboratory Session
WARNING!!! DUMMY ENTRY
I Laboratory Exercise
Lab Session 9b Expression of Fusion Protein from Positive Clones, SDS–PAGE and Western Blot, Part I
I Introduction
II Laboratory Exercises
A Sample Preparation
Gel Apparatus Preparation
B Loading Samples on the Gel
C Electrophoresis
D Stopping Electrophoresis
E Transferring to Nitrocellulose
F Disassemble and Store
Reference
Discussion Questions
Lab Session 10 Expression of Fusion Protein from Positive Clones, SDS–PAGE, and Western Blot: Part II
I Introduction
II Laboratory Exercises
A SDS–PAGE and Western Blot, Part II
Ponceau Stain
Blocking
Incubation With α-EGFP (Primary Antibody)
Incubation With Goat Anti-Mouse Peroxidase (Secondary Antibody)
Detection of Peroxidase Activity
Option A—Colorimetric Detection
Option B—Enhanced Chemiluminescence
B Replica Plate Positive Clone
Discussion Questions
Lab Session 11 Extraction of Recombinant Protein From Escherichia coli Using a Glutathione Affinity Column
Lab Session 11a Interim Laboratory Session
WARNING!!! DUMMY ENTRY
I Laboratory Exercise
Lab Session 11b Extraction of Recombinant Protein from Escherichia coli and Purification Using a Glutathione Affinity Column
I Introduction
II Laboratory Exercises
A Growing Bacterial Suspension Cultures for Fusion Protein Purification
B Harvesting IPTG-Induced Cultures
C Breaking Open Bacterial Cells
Option A—Sonication and Freeze-Thawing (Physical Method)
Option B—Bacterial Protein Extraction Reagent, B-PER (Chemical Method)
D Removing Insoluble Debris From the Crude Homogenate
E Purifying Protein by Affinity Chromatography
Discussion Questions
Lab Session 12 Analysis of Purification Fractions
Lab Session 12a Analysis of Purification Fractions
I Introduction
A Protein Quantification by Fluorescence
B Protein Quantification by Bradford Assay
II Laboratory Exercises
A Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Purified Fusion Protein
Staining the Gel
B Protein Quantification Assays
Protein Quantification Using Fluorescence Analysis
To Calculate the Concentration and Amount of Protein in Each Sample
Protein Quantification Using Bradford Protein Assay next line under subheading
Lab Session 12b Replica Plating (Only if Performing Lab Session 13)
I Laboratory Exercise
Discussion Questions
Part IV:
Analysis of mRNA Levels
Challenges of Working With RNA
Lab Session 13 Total RNA Purification
Lab Session 13a Interim Laboratory Session
WARNING!!! DUMMY ENTRY
I Laboratory Exercise
Lab Session 13b Total RNA Purification
I Introduction
II Laboratory Exercises
A Purification of Total RNA
B Sample Preparation
C DNase Digestion
D Quantification of RNA
Reference
Discussion Questions
Lab Session 14 Analysis of gst::egfp mRNA Levels by RT-qPCR: Part I
I Introduction
A Reverse Transcription
B Quantitative PCR
II Laboratory Exercises
A Reverse Transcription
B Quantitative PCR
Reference
Discussion Questions
Lab Session 15 Analysis of gst::egfp mRNA Levels by RT-qPCR: Part II
I Introduction
Step 1: Normalize to an Endogenous Reference
Step 2: Compare to a Calibrator
Step 3: Calculate Relative Quantity
II Laboratory Exercise
References
Discussion Questions
Lab Session 16 Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part I
I Introduction
II Laboratory Exercises
A Reverse Transcription
B Semiquantitative Polymerase Chain Reaction
Discussion Questions
Lab Session 17 Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part II
I Introduction
II Laboratory Exercises
A Agarose Gel Electrophoresis
B Quantification
Discussion Questions
Part V:
Modulation of Gene Expression
Time Line
Lab Session 18 Culturing Mammalian Cells
I Introduction to Sterile Technique
II Laboratory Exercises
A Operating and Using a Laminar Flow Hood
B Other Considerations: Using Incubators and Microscopes Within a Cell Culture Facility
C Counting and Plating Mammalian Cells
Reference
Discussion Questions
Lab Session 19 Transient Transfection of Mammalian Cells
Lab Session 19a Interim Laboratory Session
WARNING!!! DUMMY ENTRY
I Introduction
II Laboratory Exercise
Lab Session 19b Analysis of EGFP Expression Using Fluorescence Microscopy
I Introduction
II Laboratory Exercise
Viewing EGFP fluorescence and acquiring images for later analysis using a fluorescence microscope.
Lab Session 19c Creating Stable EGFP Expressing HEK293 Cells
I Introduction
II Laboratory Exercises
A Performing a Kill Curve
B Selecting for Stables
References
Discussion Questions
Lab Session 20 RNAi-Mediated Knockdown of EGFP: Part I
I Introduction
II Laboratory Exercise
In silico design of siRNAs targeting egfp
References
Discussion Questions
Lab Session 21
Lab Session 21 RNAi-Mediated Knockdown of EGFP: Part II
I Introduction
Lab Session 21a Counting and Plating EGFP Expressing Mammalian Cells
I Laboratory Exercise
Lab Session 21b Interim Laboratory Session
WARNING!!! DUMMY ENTRY
I Laboratory Exercise
Lab Session 22 RNAi-Mediated Knockdown of EGFP: Part III
I Introduction
II Laboratory Exercise
Discussion Questions
Lab Session 23
Lab Session 23 CRISPR-Mediated Knockout of EGFP: Part I
Lab Session 23a CRISPR-Mediated Knockout of egfp, Part I
I Introduction
II Laboratory Exercise
Lab Session 23b Interim Laboratory Session
WARNING!!! DUMMY ENTRY
I Introduction
II Laboratory Exercise
Discussion Questions
Lab Session 23c Interim Laboratory Session
WARNING!!! DUMMY ENTRY
I Introduction
II Laboratory Exercise
References
Lab Session 24 CRISPR-Mediated Knockout of EGFP: Part II
I Introduction
II Laboratory Exercise
Reference
Discussion Questions
Lab Session 25 Advanced CRISPR: Part I
I Introduction
A sgRNA Design
B Annealed Oligo Cloning
C PCR Primer Design Basics
II Laboratory Exercises
A sgRNA Design and Analysis
B Primer Design
C Restriction Enzyme Digestion and Dephosphorylation of the CRISPR Vector
D Agarose Gel Electrophoresis
E Gel Extraction of Digested CRISPR Vector Backbone
F DNA Concentration Determination
References
Discussion Questions
Lab Session 26 Advanced CRISPR: Part II
I Introduction
II Laboratory Exercises
A sgRNA Oligo Annealing and Phosphorylation
B Ligation of sgRNAs to Digested CRISPR Vector
C Transformation Into E. coli
Discussion Questions
Lab Session 19 Transient Transfection of Mammalian Cells
Lab Session 27a Interim Laboratory Session
Counting Transformants and Inoculating Cultures for Miniprep DNA
I Introduction
II Laboratory Exercise
Lab Session 27b Advanced CRISPR: Part III
I Introduction
II Laboratory Exercises
A Isolation of Miniprep DNA
B Preparation for Sequencing
Discussion Questions
Lab Session 28 Advanced CRISPR: Part IV
Lab Session 28a Advanced CRISPR: Part IV
I Introduction
II Laboratory Exercises
A Computational Analysis of DNA Sequences
B Counting and Plating EGFP Expressing Mammalian Cells
Discussion Questions
Lab Session 28b Interim Laboratory Session
WARNING!!! DUMMY ENTRY
I Introduction
II Laboratory Exercise
Lab Session 29 Advanced CRISPR: Part V
I Introduction
II Laboratory Exercises
A Harvesting Cells
B Lysis and Extraction
C Pouring Agarose Gels
D PCR
Discussion Questions
Lab Session 30 Advanced CRISPR: Part VI
I Introduction
II Laboratory Exercises
A Verifying the PCR Product
B Denaturing and Reannealing Reactions
C Enzyme Digestion
D Gel Analysis
Reference
Discussion Questions
Appendix A Equipment
Shared Equipment
Appendix B Prep List
Notes to Prep Staff
Plasmids and Escherichia coli Host Strains
Antibiotics
Aliquots
Bacterial Waste
Solid
Liquid
Autoclaving
Liquid
Biohazard Waste
Dry Goods
General Lab Preparation
Students
Supplies and Reagents
Supplies and Reagents for General Use
Recipes for General Use
LB Broth and LB Agar
IPTG (20mg/mL)
Kanamycin (30mg/mL)
Ampicillin (100mg/mL)
5× TBE Stock (Tris-Borate-EDTA)
GelRed Stock (10,000×)
DNA Primers
siRNA Oligos
Lab Session 1
Getting Oriented; Practicing With Micropipettes
Lab Session 2
Purification and Digestion of Plasmid (Vector) DNA
Lab Session 3
Completion of Vector Preparation and Polymerase Chain Reaction Amplification of egfp
Lab Session 4
Preparation of Insert DNA (egfp) PCR Product
Lab Session 5
DNA Ligation and Transformation of Escherichia coli
Lab Session 6a
Interim Laboratory Session
Counting Transformants, Replica Plating, and Inoculating Overnight Cultures
Lab Session 6b
Isolation of Miniprep DNA From Potential Transformants
Lab Session 6c
PCR Screening
Lab Session 6d
Visualization of Green Fluorescent Protein, Part I
Lab Session 7a
Restriction Digestion of DNA
Lab Session 7b
Analysis of PCR Screen and Miniprep Digestions
Lab Session 7c
Analysis of DNA Sequence From a Positive Clone, Part I
Lab Session 8
Analysis of DNA Sequence From a Positive Clone, Part II
Lab Session 9a
Interim Laboratory Session
Inoculate Culture for SDS-PAGE
Lab Session 9b
Expression of Fusion Protein From Positive Clones, SDS–PAGE, and Western Blot: Part I
Recipes
2× YT Broth
2× YT/Kan/IPTG
Tris–Glycine–SDS Running Buffer
Lab Session 10
Expression of Fusion Protein From Positive Clones, SDS–PAGE and Western Blot: Part II
Recipes
Tris-Buffered Saline (pH 7.4)
Chloronaphthol Stock Solution
Peroxide Stain
Lab Session 11a
Interim Laboratory Session
Inoculate Cultures for Protein Purification
Lab Session 11b
Extraction of Recombinant Protein From Escherichia coli Using a Glutathione Affinity Column
Recipes
Phosphate-Buffered Saline, pH 7.4
Phosphate-Buffered Saline With Pefabloc
Phosphate-Buffered Saline With 10mM Reduced Glutathione (GST Elution Buffer)
Lab Session 12a
Analysis of Purification Fractions
SDS–PAGE
Fluorescence Analysis
Bradford Protein Analysis
Lab Session 12b
Replica Plating (Only If Performing Lab Session 13)
Lab Session 13a
Interim Laboratory Session
Inoculate Cultures for RNA Purification
Lab Session 13b
Total RNA Purification
Recipes
Lactose (20mg/mL)
2× YT/Kan/IPTG
2× YT/Kan/Lac
Lysozyme (40mg/mL)
DNaseI (2.73 Kunitz Units/L)
Lab Session 14
Analysis of gst::egfp mRNA Levels by RT-qPCR: Part I
Lab Session 15
Analysis of gst::egfp mRNA Levels by RT-qPCR: Part II
Lab Session 16
Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part I
Lab Session 17
Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part II
Lab Session 18
Culturing Mammalian Cells
Recipe
Complete Medium
Cell Culture Preparation
Lab Session 19a
Interim Laboratory Session
Transient Transfection of pEGFP-N1 in HEK293 Cells Using FuGENE HD
Lab Session 19b
Analysis of EGFP Expression Using Fluorescence Microscopy
Lab Session 19c
Creating Stable EGFP-Expressing HEK293 Cells
Selecting for Stables
Lab Session 20
RNAi-Mediated Knockdown of EGFP: Part I
Lab Session 21a
RNAi-Mediated Knockdown of EGFP: Part II
Counting and Plating EGFP Expressing Mammalian Cells
Lab Session 21b
Interim Laboratory Session
Transient Transfection of siRNAs
Lab Session 22
RNAi-Mediated Knockdown of EGFP: Part III
Lab Session 23a
CRISPR-Mediated Knockout of EGFP: Part I
Lab Session 23b
Interim Laboratory Session
Transfection of CRISPR Vectors
Lab Session 23c
Interim Laboratory Session
Expansion of Transfected Cells
Lab Session 24
CRISPR-Mediated Knockout of EGFP: Part II
Lab Session 25
Advanced CRISPR: Part I
Lab Session 26
Advanced CRISPR: Part II
Lab Session 27a
Advanced CRISPR: Part III
Interim Laboratory Session
Counting Transformants and Inoculating Cultures for Miniprep DNA
Lab Session 27b
Advanced CRISPR: Part III
Lab Session 28a
Advanced CRISPR: Part IV
Lab Session 28b
Interim Laboratory Session
Transfection of CRISPR Vectors
Lab Session 29
Advanced CRISPR: Part V
Lab Session 30
Advanced CRISPR: Part VI
Appendix C Preparation of Competent Escherichia coli Cells
Introduction
Protocol
Preparation of Chemically Competent Cells by Calcium Chloride Treatment
Transformation Control
Reference
Appendix D Pre-Lab Questions
Lab Session 1
Lab Session 2
Lab Session 3
Lab Session 4
Lab Session 5
Lab Session 6
Lab Session 7
Lab Session 8
Lab Session 9
Lab Session 10
Lab Session 11
Lab Session 12
Lab Session 13
Lab Session 14
Lab Session 15
Lab Session 16
Lab Session 17
Lab Session 18
Lab Session 19
Lab Session 20
Lab Session 21
Lab Session 22
Lab Session 23
Lab Session 24
Lab Session 25
Lab Session 26
Lab session 27
Lab Session 28
Lab Session 29
Lab Session 30
Index
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