Contents
Part I Molecular Biology Techniques
Chapter 1
Structure and Function of the Human Genome
FRIEDHELM HILDEBRANDT
3
Chapter 2
Enzymatic Modification of DNA and RNA
KAI-OLAF NETZER . • • • • • . • • . • . . • • . • . • . . • • . . • . . . . . . . . • • . . . 24
Subprotocol 1: Restrietion Endonucleases . . . . . . . . . . . . . . . . . . . . . 24
Subprotocol 2: Exonucleases, Other Nucleases and Ligases . . . . . . . . 33
Subprotocol 3: Phosphatases - CIP Reaction . . . . . . . . . . . . . . . . . . . 36
Subprotocol 4: Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Subprotocol 5: DNA-Dependent DNA PolymerasesE. Coli DNA Polymerase 1 and Klenow Enzyme .... 40
Subprotocol 6: T4 and T7 DNA Polymerase . . . . . . . . . . . . . . . . . . . . 42
Subprotocol 7: RNA-Dependent DNA Polymerases .............. 44
Subprotocol8: DNA- Dependent RNA Polymerases ............ 47
Subprotocol 9: Terminal Deo:xynucleotidyl Transferase (TdT) . . . . . . 49
Chapter 3
Purification and Nucleic Acids From Eukaryotic Cells
HANS GERD NOTHW ANG AND FRIEDHELM HILDEBRANDT • • . • . • . • 50
Subprotocoll: Extraction of Nucleic Acids . . . . . . . . . . . . . . . . . . . . 50
Subprotocol 2: Isolation of Genomic DNA From Mammalian Cells . . 56
Subprotocol 3: Isolation of Cytoplasmic RNA . . . . . . . . . . . . . . . . . . 60
Subprotocol 4: Rapid Preparation of Total RNA by Acid
Guanidinium Thiocyanate/Phenol/Chloroform/
Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Subprotocol 5: Preparation of Poly(A)+ RNA . . . . . . . . . . . . . . . . . . . 67