PCR实验的实验室参考手册，PDF格式，可供实验室参考。
PREFACE.................................................................................... 4
LAB SUPPLY SELECTION ........................................................ 5
LABORATORY GLOVES ................................................................ 5
PCR MICROTUBES ..................................................................... 9
PIPETTS & TIPS ........................................................................ 12
PCR SEALING & STORAGE PRODUCTS...................................... 16
PRIMERS .................................................................................. 19
PRIMER DESIGN TIPS ................................................................ 19
REVIEWS OF ON-LINE AND FREEWARE PRIMER DESIGN TOOLS...... 22
PCR METHODS ........................................................................ 32
STANDARD PCR ....................................................................... 32
LA PCR................................................................................... 33
HOT START PCR ...................................................................... 34
TOUCHDOWN PCR.................................................................... 39
RT PCR................................................................................... 40
IN SITU PCR............................................................................. 45
QUANTITATIVE PCR.................................................................. 49
PCR VARIABLES ..................................................................... 51
Primers................................................................................. 52
Polymerases ........................................................................ 52
Templates ............................................................................ 53
Magnesium ions................................................................... 54
Preincubation Temperatures and Times.............................. 55
Melting Temperatures and Times........................................ 55
Temperature - Annealing/Hybridization ............................... 56
Temperature - Extension/Polymerization............................. 56
Estimating extension times for LA PCR............................... 57
Cycles .................................................................................. 57
Reaction Volumes................................................................ 58
PCR POLYMERASES............................................................... 60
TAQ (THERMUS AQUATICUS)...................................................... 62
KLENTAQ (THERMUS AQUATICUS, N-TERMINAL DELETION MUTANT)
..63
STOFFEL FRAGMENT ................................................................. 64
TTH (THERMUS THERMOPHILUS).................................................64
PFU (PYROCOCCUS FURIOSUS) ..................................................65
VENT (THERMOCOCCUS LITORALIS)............................................66
DEEP VENT (PYROCOCCUS SPECIES GB-D) ...............................67
ULTMA (THERMOTOGA MARITIMA)..............................................68
PCR TROUBLESHOOTING......................................................69
Non-Specific Product Yields.................................................69
Little or No Product Yield......................................................70
Multiple Product Yields or High Molecular-Weight Smear is
Observed..............................................................................72
Primer-Dimers ......................................................................73
About Inhibitors.....................................................................74
About Enhancers..................................................................74
How to Reduce Contamination.............................................76
Techniques Used to Prevent Contamination........................77
PCR INHIBITORS & ENHANCERS ..........................................79
GENERAL/INTRO, FECES, FORMALIN-FIXED PARAFFIN SAMPLE ...79
FECES, NORWALK VIRUS, ROTAVIRUS, URINE ............................82
RESPIRATORY FLUID, BLOOD, SHELLFISH...................................86
BODY FLUIDS AND FOOD............................................................90
PLANTS & SOIL .........................................................................94
SPECIAL PCR TOPICS.............................................................97
WHY AREN'T PCR-BASED DIAGNOSTICS MAIN STREAM?............97
PCR-BASED DETECTION KITS PART I.......................................100
PCR-BASED DETECTION KITS PART II.....................................103
FISH, PRINS & CYTOGENICS: DO YOU HAVE A LIGHT? ..........106
CLINICAL APPLICATIONS OF DOT BLOTS ...................................110
APPENDIX A ¨C UNITS AND FORMULAS..............................113
APPENDIX B ¨C THE GENETIC CODE....................................116
APPENDIX C ¨C ONLINE GENOMIC DATABASES................117
APPENDIX D ¨C PCR PRODUCT SUPPLIERS .......................124
APPENDIX E ¨C ONLINE BIOSUPPLIERS..............................137
APPENDIX F - CONTRIBUTORS ...........................................139
INDEX
