Biochemistry and Molecular Biology Laboratory Course

Part 1 Basic techniques of biochemistry and molecularbiology
Chapter 1 Basic techniques of preparation of biomacromolecules 1
Section 1 Salting out 2
Section 2 Dialysis and ultrafiltration 3
Chapter 2 Spectrophotometry 5
Section 1 Basic principles 5
Section 2 The basic construction of spectrophotometers 7
Section 3 Some home-made spectrophotometers 9
Chapter 3 Chromatography 12
Section 1 Adsorption chromatography 12
Section 2 Partition chromatography 12
Section 3 Ion-exchange chromatography 14
Section 4 Gel filtration 17
Section 5 Affinity chromatography 18
Chapter 4 Electrophoretic techniques 20
Section 1 Basic principles 20
Section 2 Several types of commonly used electrophoresis 23
Chapter 5 Centrifugation techniques 29
Section 1 Basic principle 29
Section 2 General description of centrifugation 30
Chapter 6 Genetic engineering and analytical technology of gene expression 34
Section 1 Genetic engineering 34
Section 2 Polymerase chain reaction 40
Section 3 Nucleic acid hybridization 45
Section 4 DNA sequencing technology 46
Part 2 Experiments of the biochemistry and molecular biology
Chapter 7 Analysis of purification and physicochemical properties of proteins 49
Experiment 1 Colorimetric methods for protein determination 49
Experiment 2 Isolation and purification of serum γ-globulin 51
Experiment 3 Cellulose acetate membrane electrophoresis of serum proteins 53
Experiment 4 Determination of relative molecular mass of protein in SDS-PAGE 55
Chapter 8 Kinetics of enzyme-catalysed reactions 58
Experiment 1 The specificity of enzyme and effect of temperature, pH on the enzyme
Activity 58
Experiment 2 Isolation of alkaline phosphatase and determination of its specific activity 60
Experiment 3 Effects of substrates concentration of enzymatic activity: determination
of Km of alkaline phosphatase 64
Experiment 4 Inhibition effect of phosphate on alkaline phosphatase 66
Experiment 5 Analysis of lactic dehydrogenase isoenzyme 67
Chapter 9 Substance metabolism 70
Experiment 1 Extraction and identification of liver glycogen 70
Experiment 2 Determination of alanine transaminase activity (Reitman-Frankels’method) 71
Experiment 3 Enzyme catalyzed transamination of amino acids detected by paper
chromatography 73
Chapter 10 Basic experiment of molecular biology 76
Experiment 1 Preparation of the genomic DNA in the eukaryotic cells 76
Experiment 2 Determination of the concentration and purity of DNA by spectroscopy 77
Experiment 3 Agarose gel electrophoresis of nucleic acids 77
Experiment 4 Polymerase chain reaction (PCR) 79
Experiment 5 Isolation and purification of the DNA fragments 80
Experiment 6 DNA ligation—constructing recombinant plasmids 82
Experiment 7 Preparation of E.coli competent cells 83
Experiment 8 Transformation of Ca2+ competent cells 84
Experiment 9 The screening of recombinant DNA 85
Experiment 10 Minipreparation of plasmid DNA 86
Experiment 11 Digestion of plasmid DNA with restriction endonucleases 89
Experiment 12 Southern blot 90
Experiment 13 Expression of exogenous gene in prokaryotic cells 93
References 95
Appendix 96
Appendix Ⅰ The calculation table of ammonium sulfate saturation 96
Appendix Ⅱ Common buffer solution preparation 96
Appendix Ⅲ The relative molecular mass and pI of protein 99
Appendix Ⅳ Common data of chromatography 101
Appendix Ⅴ The conversion between centrifugation speed of centrifugation force 102