Alkami Quick Guide™ for PCR |
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www.Alkami.org |
Alkami Quick Guide™ |
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Alkami Quick Guide™ |
for PCR |
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for PCR |
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A laboratory reference for the |
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A laboratory reference for the |
Polymerase Chain Reaction |
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Polymerase Chain Reaction |
For updates, links, and resources, visit us at http://www.Alkami.org
Copyright © 1999 by Alkami Biosystems, Inc.
All Rights Reserved
Printed in the United States of America
November 1999
Volume I |
First Edition |
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Although every effort has been made to ensure the correctness of the information in this booklet, the publisher and authors do not assume, and hereby disclaim, any liability to any party for any loss or damage caused by errors, omissions, misleading information, or any potential problem caused by information in this guide, even if such errors or omission are a result of negligence, accident, or any other cause.
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Alkami Quick Guide™ for PCR
Table of Contents
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) |
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STOFFEL FRAGMENT ................................................................. |
64 |
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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 |
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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 – UNITS AND FORMULAS .............................. |
113 |
APPENDIX B – THE GENETIC CODE.................................... |
116 |
APPENDIX C – ONLINE GENOMIC DATABASES................ |
117 |
APPENDIX D – PCR PRODUCT SUPPLIERS ....................... |
124 |
APPENDIX E – ONLINE BIOSUPPLIERS.............................. |
137 |
APPENDIX F - CONTRIBUTORS ........................................... |
139 |
INDEX |
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