Molecular Biology Techniques. A Classroom Laboratory Manual

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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