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