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Subcloning Notebook
Subcloning is a basic molecular biology procedure used to move inserts from one vector to another. Essentially all subcloning reactions proceed as follows: You release and purify your insert from the parent vector, ligate the insert into a prepared destination vector, transform the ligation reaction into competent bacterial cells, then screen the transformed cells for the insert. The Subcloning Notebook will lead you through every step in this process.
Table of Contents Page
Chapter 1: Classic Subcloning (.pdf, 845kb)
Basic Steps for Subcloning
Subcloning Strategy
Restriction Digestion
Double Enzyme Digests
Partial Restriction Digestion
Creating Blunt Ends
Dephosphorylating Vectors
Ligation
Purifying Vector and Insert
Gel Electrophoresis
DNA Markers
Ordering Information 3
Chapter 2: PCR Subcloning (.pdf, 488kb)
Introduction
T-Vector Systems
Giving Blunt-Ended DNA an A-Tail for T-Vector Subcloning
Subcloning with RE Sites
Subcloning using PCR Primers Containing Restriction Sites
Ordering Information 35
Chapter 3: Transforming Bacteria (.pdf, 366kb)
Properties of E. coli Strains for Subcloning
Ready-to-Use Competent Cells
Determining Transformation Efficiency of Competent Cells
Transforming Ligation Reactions
Media and Solutions 43
Chapter 4: Screening for Recombinants (.pdf, 431kb)
Introduction
Colony PCR
Go Directly to Gel
Screening by Plasmid Minipreps and RE Digests
Plasmid Minipreps
Troubleshooting Subcloning Experiments
Ordering Information 49
Chapter 5: Technical Appendix (.pdf, 274kb)
Restriction Enzyme Activity in 10X Buffers, Reaction Temperature
and Heat Inactivation
Isoschizomers
Compatible Ends
Site-Specific Methylation Sensitivity of Promega Restriction Enzymes
Restriction Enzyme Buffer Composition
Copy Number of Commonly Used Plasmids
Star Activity
Genotypes of Frequently Used Bacterial Strains
Genetic Markers in E. coli
Nucleic Acid Calculations
Formulas for DNA Molar Conversions
http://www.promega.com/guides/subcloning_guide/Subcloning_ntbk.pdf
Beginning Molecular Biology Laboratory Gudie
新手入门的好教材，非常全面，非常亲切。值得收藏。
http://www.research.umbc.edu/~jwolf/method1.html
内容介绍：
CHAPTER 1: General Laboratory Methods
Safety Procedures
Preparation of Solutions
Disposal of Buffers and Chemicals
Equipment Micropipets
Using a pH meter
Autoclave operating procedures
Operating instructions for spectrophotometer
Working with DNA
Sterile Technique
CHAPTER 2: Instructions for Notebook Keeping
CHAPTER 3: Computer User's Guide
UNIX commands
File Transfer
The World Wide Web
The Wisconsin Package - gcg.
CHAPTER 4: Molecular Biology Methods
M.1: Preparation of genomic DNA from bacteria
M.2: PCR amplification of DNA
M.3: Restriction enzyme digestion of DNA
M.4: Phenol/chloroform extraction of DNA
M.5: Ethanol precipitation of DNA
M.6: Agarose gel electrophoresis
M.7: Transformation of E. coli by electroporation
M.8: Wizard PCR preps DNA purification system
M.9: Alternate method for purifying DNA from agarose gels
M.10: Transfection of mammalian cells using Lipofectamine (LTI)
M.11: Southern blotting
M.12: RT-PCR Protocol
M.13: Preparation of sequencing gels
M.14: Isolation of RNA from mammalian cells using RNAZOL (Teltest)
CHAPTER 5: Tissue Culture Methods
Types of cells grown in culture
Work area and equipment
Preservation and storage
Maintenance
Safety considerations
Tissue culture methods
Determining cell counts
Cloning Enzymes
Cloning Enzymes, one in the Enzyme Resource Guide series, highlights those enzymes important in nucleic acid cloning procedures. Enzymes that modify nucleic acids provide the foundation for many molecular biology techniques: enzymes that are used to synthesize, degrade, join or remove portions of nucleic acids in a controlled and generally defined manner. Specific features of the in vivo functions of these enzymes have been exploited in vitro to provide many of the protocols currently used in nucleic acid manipulations.This guide mainly is targeted Ligases, Kinases and Phosphatases.
Table of Contents Page
Enzyme Activities Diagram
The Cloning Enzymes
Gene Cloning
Ligases
Kinases and Phosphatases
RecA Protein and AgarACE(r) Enzyme
Technical Appendix
http://www.promega.com/guides/cloning_guide/cloningenz.pdf
Nucleic Acid Purification Systems
The isolation and purification of DNA is crucial to many applications in molecular biology and techonogy. Over the past decade, the purification of DNA has evolved from a multi-step process involving organic chemicals to a much simpler process, that is safer and faster. Promega continues to develop new DNA purification products to meet the diverse and changing needs of today's life science researcher. This Nucleic Acid Purification Systems guide presents our many new and innovative products for use in DNA and RNA purification.
Table of Contents Page
Genomic DNA
Plasmid DNA
Fragment DNA
Total RNA
http://www.promega.com/guides/nadp_guide/napd_guide.pdf
RNA Analysis Notebook
RNA analysis is the starting point for many molecular biology procedures. The RNA Analysis Notebook provides an introduction to RNA procedures such as purifying RNA, amplifying via RT-PCR, making RNA in vitro and analyzing RNA by microarray. In addition, the RNA Analysis Notebook highlights products for performing these procedures.
Table of Contents
Working with RNA
Purifying RNA and mRNA
Amplifying RNA with RT-PCR
Analyzing RNA with Microarrays
Making RNA in vitro
Silencing RNA in vivo (RNAi)
http://www.promega.com/guides/rna_guide/rna_gde.pdf
DNA Analysis Notebook
DNA analysis is the starting point for many molecular biology procedures. The DNA Analysis Notebook provides an introduction to DNA procedures such as purifying genomic DNA, amplifying via PCR, retrieving DNA from PCR and cloning PCR DNA. In addition, the DNA Analysis Notebook features Promega products for performing these procedures.
Contents
Table of Contents
Genomic DNA Purification
Amplifying DNA
PCR Clean-Up
Cloning PCR DNA
DNA Analysis Tools
http://www.promega.com/guides/dna_guide/dna_guide.pdf
Subcloning is a basic molecular biology procedure used to move inserts from one vector to another. Essentially all subcloning reactions proceed as follows: You release and purify your insert from the parent vector, ligate the insert into a prepared destination vector, transform the ligation reaction into competent bacterial cells, then screen the transformed cells for the insert. The Subcloning Notebook will lead you through every step in this process.
Table of Contents Page
Chapter 1: Classic Subcloning (.pdf, 845kb)
Basic Steps for Subcloning
Subcloning Strategy
Restriction Digestion
Double Enzyme Digests
Partial Restriction Digestion
Creating Blunt Ends
Dephosphorylating Vectors
Ligation
Purifying Vector and Insert
Gel Electrophoresis
DNA Markers
Ordering Information 3
Chapter 2: PCR Subcloning (.pdf, 488kb)
Introduction
T-Vector Systems
Giving Blunt-Ended DNA an A-Tail for T-Vector Subcloning
Subcloning with RE Sites
Subcloning using PCR Primers Containing Restriction Sites
Ordering Information 35
Chapter 3: Transforming Bacteria (.pdf, 366kb)
Properties of E. coli Strains for Subcloning
Ready-to-Use Competent Cells
Determining Transformation Efficiency of Competent Cells
Transforming Ligation Reactions
Media and Solutions 43
Chapter 4: Screening for Recombinants (.pdf, 431kb)
Introduction
Colony PCR
Go Directly to Gel
Screening by Plasmid Minipreps and RE Digests
Plasmid Minipreps
Troubleshooting Subcloning Experiments
Ordering Information 49
Chapter 5: Technical Appendix (.pdf, 274kb)
Restriction Enzyme Activity in 10X Buffers, Reaction Temperature
and Heat Inactivation
Isoschizomers
Compatible Ends
Site-Specific Methylation Sensitivity of Promega Restriction Enzymes
Restriction Enzyme Buffer Composition
Copy Number of Commonly Used Plasmids
Star Activity
Genotypes of Frequently Used Bacterial Strains
Genetic Markers in E. coli
Nucleic Acid Calculations
Formulas for DNA Molar Conversions
http://www.promega.com/guides/subcloning_guide/Subcloning_ntbk.pdf
Beginning Molecular Biology Laboratory Gudie
新手入门的好教材，非常全面，非常亲切。值得收藏。
http://www.research.umbc.edu/~jwolf/method1.html
内容介绍：
CHAPTER 1: General Laboratory Methods
Safety Procedures
Preparation of Solutions
Disposal of Buffers and Chemicals
Equipment Micropipets
Using a pH meter
Autoclave operating procedures
Operating instructions for spectrophotometer
Working with DNA
Sterile Technique
CHAPTER 2: Instructions for Notebook Keeping
CHAPTER 3: Computer User's Guide
UNIX commands
File Transfer
The World Wide Web
The Wisconsin Package - gcg.
CHAPTER 4: Molecular Biology Methods
M.1: Preparation of genomic DNA from bacteria
M.2: PCR amplification of DNA
M.3: Restriction enzyme digestion of DNA
M.4: Phenol/chloroform extraction of DNA
M.5: Ethanol precipitation of DNA
M.6: Agarose gel electrophoresis
M.7: Transformation of E. coli by electroporation
M.8: Wizard PCR preps DNA purification system
M.9: Alternate method for purifying DNA from agarose gels
M.10: Transfection of mammalian cells using Lipofectamine (LTI)
M.11: Southern blotting
M.12: RT-PCR Protocol
M.13: Preparation of sequencing gels
M.14: Isolation of RNA from mammalian cells using RNAZOL (Teltest)
CHAPTER 5: Tissue Culture Methods
Types of cells grown in culture
Work area and equipment
Preservation and storage
Maintenance
Safety considerations
Tissue culture methods
Determining cell counts
Cloning Enzymes
Cloning Enzymes, one in the Enzyme Resource Guide series, highlights those enzymes important in nucleic acid cloning procedures. Enzymes that modify nucleic acids provide the foundation for many molecular biology techniques: enzymes that are used to synthesize, degrade, join or remove portions of nucleic acids in a controlled and generally defined manner. Specific features of the in vivo functions of these enzymes have been exploited in vitro to provide many of the protocols currently used in nucleic acid manipulations.This guide mainly is targeted Ligases, Kinases and Phosphatases.
Table of Contents Page
Enzyme Activities Diagram
The Cloning Enzymes
Gene Cloning
Ligases
Kinases and Phosphatases
RecA Protein and AgarACE(r) Enzyme
Technical Appendix
http://www.promega.com/guides/cloning_guide/cloningenz.pdf
Nucleic Acid Purification Systems
The isolation and purification of DNA is crucial to many applications in molecular biology and techonogy. Over the past decade, the purification of DNA has evolved from a multi-step process involving organic chemicals to a much simpler process, that is safer and faster. Promega continues to develop new DNA purification products to meet the diverse and changing needs of today's life science researcher. This Nucleic Acid Purification Systems guide presents our many new and innovative products for use in DNA and RNA purification.
Table of Contents Page
Genomic DNA
Plasmid DNA
Fragment DNA
Total RNA
http://www.promega.com/guides/nadp_guide/napd_guide.pdf
RNA Analysis Notebook
RNA analysis is the starting point for many molecular biology procedures. The RNA Analysis Notebook provides an introduction to RNA procedures such as purifying RNA, amplifying via RT-PCR, making RNA in vitro and analyzing RNA by microarray. In addition, the RNA Analysis Notebook highlights products for performing these procedures.
Table of Contents
Working with RNA
Purifying RNA and mRNA
Amplifying RNA with RT-PCR
Analyzing RNA with Microarrays
Making RNA in vitro
Silencing RNA in vivo (RNAi)
http://www.promega.com/guides/rna_guide/rna_gde.pdf
DNA Analysis Notebook
DNA analysis is the starting point for many molecular biology procedures. The DNA Analysis Notebook provides an introduction to DNA procedures such as purifying genomic DNA, amplifying via PCR, retrieving DNA from PCR and cloning PCR DNA. In addition, the DNA Analysis Notebook features Promega products for performing these procedures.
Contents
Table of Contents
Genomic DNA Purification
Amplifying DNA
PCR Clean-Up
Cloning PCR DNA
DNA Analysis Tools
http://www.promega.com/guides/dna_guide/dna_guide.pdf
