Protocols
• Primer on Cloning, Expression, and Purification
[Under
Construction] This set
of protocols is meant to aid a new student just starting to learn molecular
biology and protein chemistry with a detailed step-wise procedure for
cloning a gene into a pET-style T7-inducible expression vector, confirming
the cloning, expressing the His•Tag protein, and purifying the protein
using metal-affinity chromatography. Standard validation and characterization
of the expressed protein is also described.
1
Cloning
[.ppt revised 2004.12.13 by G.Yuan]
1.1 Vector Selection
[.doc revised
2005.09.06 by M.Murugesan]
1.2 Primer Design
[.doc revised
2005.09.06 by M.Murugesan]
1.3 PCR Amplification
of Gene [.pdf
revised 2004..11.09 by G.Lipscomb; .doc
revised 2005.09.06 by M.Murugesan]
1.3.1 Gradient
PCR
1.4 Ligation
1.4.1 PCR
Product Purification and Restriction [.doc
revised 2005.09.06 by M.Murugesan]
1.4.2 Vector
Restriction and Dephosphorylation
1.4.3 Vector
Gel Purification
1.4.4 Ligation
1.5 Transformation
for Plasmid Replication
1.5.1 Choice
of E. coli Strain
1.5.2 Competent
Cells
1.5.3 Transformation
and Colony Selection [.doc
revised 2005.09.06 by M.Murugesan]
1.6 Plasmid
Purification [.doc
revised 2005.09.06 by M.Murugesan]
1.7 Verification
of Insert
1.7.1 Restriction
Mapping
1.7.2 Sequence
Verification
2
Expression
2.1 Transformation
for Expression
2.1.1 Choice
of E. coli Strain [.doc
revised 2005.08.24 by G.Lipscomb]
2.1.2 Competent
Cells [.doc
revised 2005.08.24 by G.Lipscomb]
2.1.3 Transformation
and Colony Selection [.doc
revised 2005.08.24 by G.Lipscomb]
2.2 Small-scale
Culture Growth for Protein Expression
2.2.1 Expression
Verification [.doc
revised 2005.08.24 by G.Lipscomb]
2.2.2 Optimization
of Expression [.doc
revised 2005.08.24 by G.Lipscomb]
2.3 Large-scale
Culture Growth for Protein Expression [.doc
revised 2005.08.24 by G.Lipscomb]
3
Purification
3.1 Preparation
of Soluble Cell Extract (for soluble protein)
3.2 Solubilization
of Inclusion Bodies (for insoluble protein)
3.3 Ni-Affinity
Chromatography for His•Tag Proteins [.doc
revised 2006.07.13 by M.Murugesan]
3.4 Buffer Exchange/Desalting
3.5 Measures
of Protein Purity
3.6 Other Separation
Techniques [.ppt
revised 2005.07.12 by M.Murugesan]
3.7 Protein
Concentration
3.8 Protein
Quantification [.ppt
revised 2005.05.10 by X.Li]
3.9 Metal/Protein
Ratio (for metalloproteins)
3.10 Protein
Storage
scott@chem.uga.edu
Department of Chemistry
University of Georgia
Athens, GA 30602-2556
706 542-2240 | FAX: 706
542-2295 |
