Gerstein, Alan S, Amersham Pharmacia Biotech, Piscataway, N, Mbproblemsolver@earthlink.net,alangerstein@am.apbiotech.com Haidaris, Constantine G, University of Rochester School of Medicine and Dentistry, Rochester, NY Herzer, sibylle, Amersham Pharmacia Biotech, Piscataway, N, sibylleherzer@am.apbiotech.com Kennedy, Michele A, Brinkmann Instruments, Inc, Westbury, NY, info(@brinkmann.com Kirkpatrick, Robert, GlaxoSmithKline, King of Prussia, PA Kracklauer. Martin. Ambion Inc. Austin TX Krueger, Gregory, Amersham Pharmacia Biotech, Piscataway, N bermoeller, Dawn Ambion Inc, Austin TX Marcy, Alice, Merck Research Labs, Rahway, N, alice_marcy@merck.com Martin,LoriA.,Ambion,Inc,Austin,Tx,moinfo@ambion.com Pfannkoch, Edward A, Gerstel Corporation, Baltimore, MD Prasauckas, Kristin A Packard Bioscience, Meriden, CT, kprasauckas(@packardinst.com Riis, Peter, Chicago, IL Robinson, Derek, New England Biolabs, Beverly, MA Shatzman, Alan R,, GlaxoSmithKline, King of Prussia, PA Smith, Tiffany ], Ambion, Inc, Austin, TX Stevens, Jane, Thermo Orion, Beverly, MA, Domcsl@thermoorion.com Trill, John., GlaxoSmithKline, King of Prussia, PA Troutman, Trevor, Sartorius Inc, Edgewood, NY Contributors
Gerstein,Alan S., Amersham Pharmacia Biotech, Piscataway, NJ, Mbproblemsolver@earthlink.net, alan.gerstein@am.apbiotech.com Haidaris, Constantine G., University of Rochester School of Medicine and Dentistry, Rochester, NY Herzer, Sibylle, Amersham Pharmacia Biotech, Piscataway, NJ, sibylle.herzer@am.apbiotech.com Kennedy, Michele A., Brinkmann Instruments, Inc., Westbury, NY, info@brinkmann.com Kirkpatrick, Robert, GlaxoSmithKline, King of Prussia, PA Kracklauer, Martin, Ambion, Inc., Austin, TX Krueger, Gregory, Amersham Pharmacia Biotech, Piscataway, NJ Obermoeller, Dawn, Ambion, Inc., Austin, TX Marcy,Alice, Merck Research Labs, Rahway, NJ, alice_marcy@merck.com Martin, Lori A., Ambion, Inc., Austin, TX, moinfo@ambion.com Pfannkoch, Edward A., Gerstel Corporation, Baltimore, MD Prasauckas, Kristin A., Packard Bioscience, Meriden, CT, kprasauckas@packardinst.com Riis, Peter, Chicago, IL Robinson, Derek, New England Biolabs, Beverly, MA Shatzman,Alan R., GlaxoSmithKline, King of Prussia, PA Smith,Tiffany J., Ambion, Inc., Austin, TX Stevens, Jane, Thermo Orion, Beverly, MA, Domcsl@thermoorion.com Trill, John J., GlaxoSmithKline, King of Prussia, PA Troutman,Trevor, Sartorius Inc., Edgewood, NY xii Contributors
Tyre, Tom, Pierce Milwaukee, Milwaukee, WI Tom.tyre@piercenet.com Volny, william R.]. Jr, Amersham Pharmacia Biotech, Piscataway, N Walsh, Paul R, New England Biolabs, Beverly, MA Contributors
Tyre,Tom, Pierce Milwaukee, Milwaukee, WI, Tom.tyre@piercenet.com Volny,William R. J. Jr.,Amersham Pharmacia Biotech, Piscataway, NJ Walsh, Paul R., New England Biolabs, Beverly, MA Contributors xiii
Index Azo measurements, 108 of spectrophotometers, 96-97 A230 measurements, 103 98-100.101-106 Achilles'heel cleavage(AC), 252 monitoring DNA purification, in genome digests, 252-255 190-191 Acid dissociation constant(K,), 33 in monitoring RNA purification, 202 Acids. See also Strong acids; Weak r polymerase chain reactions, 312 buffer absorption of, 38 Azo measurements, 103, 104, 105-106, buffering of, 32 108,276-277,280 as buffers. 33 ariations among, 278 Acid-type buffers, amine-type buffers Azso measurements, 103, 105-106, 108 versus, 32 Acknowledging orders, 19 Agoo measurements, 102 acrylamide Ago measurements, 280 as neurotoxin. 335 Absorbance(A) polymerization of, 338, 339, 341-343 calculating, 275-276 pore size with, 339-341 concentration and. 104-105.108- safe disposal of, 335-336 safety issues with, 334-336 nucleotide 272-273. 275 shelf life of, 336 optical density versus, 275-277 of,33 th le Acrylamide gel solutions, degassing Absorbance accuracy, of spectrophotometers, 99, 102-103, Acrylamide 103-104 Acrylamido with gradient Absorbance range, in gels, 346 spectrophotometry, 105-106 crylic plastic, as shielding, 163 Absorbance ratio. Active nucleic acid transfer. 418 spectrophotometry, 103, It Activity tables for restriction 105-106 Absorbance unit(AU), 276-27 Additives in polymerase chain reactions, 306, Absorption coefficient, 277 Absorption maxima, of nucleotides for protein solubility, 355 (table), 272 Adenine methylases, in genomic Absorptivity, 277 digests, 250-252 Accidental self-inoculation. in Adeno-associated virus(AAD),in biosafety, 123 eukaryotic expression, 50 Accuracy. See also Calibration Adenoviruses, in eukaryotic of pH meters, 87-89, 90 03-504 of pipettes, 75 Adjustable-volume pipettes, 67-68 543
543 A210 measurements, 108 A230 measurements, 103 A260 : A280 ratio in monitoring DNA purification, 190–191 in monitoring RNA purification, 202 for polymerase chain reactions, 312 in spectrophotometry, 106 A260 measurements, 103, 104, 105–106, 108, 276–277, 280 variations among, 278 A280 measurements, 103, 105–106, 108 A320 measurements, 104 A600 measurements, 102 A820 measurements, 280 Absorbance (A) calculating, 275–276 concentration and, 104–105, 108– 109 nucleotide, 272–273, 275 optical density versus, 275–277 path length and, 286 Absorbance accuracy, of spectrophotometers, 99, 102–103, 103–104 Absorbance range, in spectrophotometry, 105–106 Absorbance ratio, in spectrophotometry, 103, 104, 105–106 Absorbance unit (AU), 276–277 Absorbency index, 277 Absorption coefficient, 277 Absorption maxima, of nucleotides (table), 272 Absorptivity, 277 Accidental self-inoculation, in biosafety, 123 Accuracy. See also Calibration of pH meters, 87–89, 90 of pipettes, 75 of spectrophotometers, 96–97, 98–100, 101–106 Achilles’ heel cleavage (AC), 252 in genome digests, 252–255 Acid dissociation constant (Ka), 33 Acids. See also Strong acids; Weak acids buffer absorption of, 38 buffering of, 32–33 as buffers, 33 Acid-type buffers, amine-type buffers versus, 32 Acknowledging orders, 19 Acrylamide as neurotoxin, 335 polymerization of, 338, 339, 341– 343 pore size with, 339–341 safe disposal of, 335–336 safety issues with, 334–336 shelf life of, 336 storage of, 336 Acrylamide gel solutions, degassing of, 371 Acrylamide poisoning, 335 Acrylamido buffers, with gradient gels, 346–347 Acrylic plastic, as shielding, 163 Active nucleic acid transfer, 418 Activity tables, for restriction enzymes, 237–238 Additives in polymerase chain reactions, 306, 307 for protein solubility, 355 Adenine methylases, in genomic digests, 250–252 Adeno-associated virus (AAD), in eukaryotic expression, 504 Adenoviruses, in eukaryotic expression, 503–504 Adjustable-volume pipettes, 67–68 Index
Advantages of immobilized Ammonium compounds, as gradient gels, 347 disinfectants, 132 clalms Ammonium ions. in dna Aeration, in gene expression, 4 Affinity purification, problems after, Ampholytes, with gradient gels, Affinity resins, problems with Amplification Affinity tags, with fusion syste in eukaryotic express 472-474.474475 in Western blotting, 387-388 Affinity techniques Analysis date, for radioisotopes, 148 in plasmid purification, 183-184 Analytical balances, 51 condary reagents Angle, of centrifuge rotors, 58-61 ar media, handling 137-138 Anhydrous buffer salts, 35 Agarose, 355 animals DNA isolation from, 188, 190 biosafety with, 128-130 for pulsed field electrophoresis, disposal of parts of infected, 246.247 129-130 RNA isolation from 203 Anion exchange(AIX) Agarose electrophoresis, 355-357 in dNA extraction. 178-179 Agarose gels, nucleic acid transfer plasmid purification, 1 from,418-420 Annular pH electrode, 80 Agarose microbeads, 251-252 Antibiotics, in eukaryotic expression Agarose plugs, 251 Antibodies ging of glassware, 138-139 gene expression and, 477 Air buoyancy, as affecting balance stripping and reprobing and Air currents, as affecting balance for Western blotting. 378 381-382 accuracy, 53 Air displacement pipette, 6 Anticoagulants, in DNA purification, Alcohols 70-171 in complex digests, 240-241 Aoyagi, Kazuo, 291 s disinfectants. 1 Applications, for Alkali fixation, crosslinking via, 423 Aspergillus, safe handling of, 127 Alkaline lysis, in plasmid purification, Aspiration, with pipettes, 68, 74, 77 Assays Alkaline phosphatase(AP) of eukaryotic expression, 515-517 for detecting proteins, 376, 377 rotein quantitation problems with, 394 Attitude, in minimizing radiation Alkaline transfer exposure, 162 conditions for 419 Authorized users, of radioisotopes. crosslinking via, 423 Aluminum foil as autoclave Autobuffer recognition, in pH met wrapping, 134 American Type Culture Collection, Autocalibration, of pH meters, 82-83 autoclave bags, in waste Amine-type buffers, acid-type buffers decontamination. 140 absorbance data and concentration agar media in, 13/-30, Op Autoclaves. See also sterilizati Amino acid carbohydrates in, 139 and,108-109 condensation with. 135 470.475 do-it-yourself, 137-140
544 Index Advantages of immobilized pH gradient gels, 347 Advertising claims, 13–14 Aeration, in gene expression, 478 Affinity purification, problems after, 485 Affinity resins, problems with, 484 Affinity tags, with fusion systems, 472–474, 474–475 Affinity techniques in plasmid purification, 183–184 secondary reagents and, 385 Agar media, handling, 137–138 Agarose, 355 DNA isolation from, 188, 190 for pulsed field electrophoresis, 246, 247 RNA isolation from, 203 Agarose electrophoresis, 355–357 Agarose gels, nucleic acid transfer from, 418–420 Agarose microbeads, 251–252 Agarose plugs, 251 Agarose preparations, table of, 356 Aging of glassware, 138–139 Air buoyancy, as affecting balance accuracy, 52 Air currents, as affecting balance accuracy, 53 Air displacement pipette, 67 Alcohols in complex digests, 240–241 as disinfectants, 131 Alkali fixation, crosslinking via, 423 Alkaline lysis, in plasmid purification, 180–182 Alkaline phosphatase (AP) for detecting proteins, 376, 377 problems with, 394 Alkaline transfer conditions for, 419 crosslinking via, 423 Aluminum foil, as autoclave wrapping, 134 American Type Culture Collection, 117 Amine-type buffers, acid-type buffers versus, 32 Amino acids absorbance data and concentration and, 108–109 in proteins, 470, 475 Ammonium compounds, as disinfectants, 132 Ammonium ions, in DNA purification, 170 Ampholytes, with gradient gels, 346–347 Amplification in eukaryotic expression, 508–509 in Western blotting, 387–388 Analysis date, for radioisotopes, 148 Analytical balances, 51 Angle, of centrifuge rotors, 58–61 Anhydrous buffer salts, 35 Animals biosafety with, 128–130 disposal of parts of infected, 129–130 Anion exchange (AIX) in DNA extraction, 178–179 in plasmid purification, 181 Annular pH electrode, 80 Antibiotics, in eukaryotic expression, 512–513 Antibodies gene expression and, 477 stripping and reprobing and, 388–389 for Western blotting, 378, 381–382, 383–384 Anticoagulants, in DNA purification, 170–171 Aoyagi, Kazuo, 291 Applications, for products, 13 Aspergillus, safe handling of, 127 Aspiration, with pipettes, 68, 74, 77 Assays of eukaryotic expression, 515–517 protein quantitation, 109–110 Attitude, in minimizing radiation exposure, 162 Authorized users, of radioisotopes, 144 Autobuffer recognition, in pH meters, 82–83 Autocalibration, of pH meters, 82–83 Autoclave bags, in waste decontamination, 140 Autoclaves. See also Sterilization agar media in, 137–138, 139 carbohydrates in, 139 condensation with, 135 do-it-yourself, 137–140
glassware in, 138-139 Baking membranes, 422 indicator tape with, 135 Balances 51-55 microbial contamination and. 124 calibrating. 55 plastic materials in, 135 factors affecting accuracy of, 51-55 safety and, 120, 139 pipette testing, 72-7 sterilizing membranes in. 417 selecting, 54-55 time requirements for, 135 service in waste decontamination, 139-140 types of, 51 wrapping for, 134 Banana plugs, in electrophoresis, 337 Autoclave settings, 133-134 Bands Autographa califomica genome, 503 after affinity purification, 485-486 Automatic temperature compensation fuzzy, 35 (ATC), in pH meters, 84-85 in native PAGE. 348 Autoradiography film, in nucleic acid with pre-stained pi hybridization. 436-441 problems with, 396 Avidin reproducible, 353 in hybridization buffer, 429 from skin keratin. 368 problems with, 395 BandShift Kit Instruction Manual in Western blotting, 381-382 (Hennighausen Lubon ), 35 386-387 Bandwidth resolution of Axenic cultures, for protozoa, 128 spectrophotometers, 97-98 Barometric pressure, in testing Baby hamster kidney(BHK)cell es,76 lines, eukaryotic expression with, Baseline flatness, of spectrophotometers, 99-100 BAC(bis-acrylylcystamine),as Bases. See also strong bases crosslinker. 338 buffer absorption of, 38 Background noise, with storage buffering of, 32-33 phosphor imagers, 447 Batch binding in rna Background stain, as problem, 362, 395-396 Batch numbers, for products, 25 Backup cultures, for experiments, 124 BCA assay, 109 Bacteria Bead milling, cell disruption via, 217-218 disruption of, 217-218 Beckman-Coulter rotor for g degradation of rNA centrifuges, 63, 6. from,215,217-218 Beer-Lambert law. 97.100. 104-105 restriction enzymes from, 227 108,275,277-278,279 safe handling of, 126-127 Bell. Peter A. 461 total RNA isolation from, 208-209 Below balance weighing, 53 Bacterial strains, maintaining, 125-126 Benzene, radioisotopes in, 147 Bacteriophages, safe handling of, 127 Bequerel(Bq), 145-146n, 155 Baculovirus. 521-532 Beta emitters implementing experiment with autoradiography film and, 438439 527-530 shielding for, 163 insect cell line versus 523 Big companies, 12-13 planning experiment with, 521-527 Binding capacity, of hybridization selecting insect cell system and membranes. 414 525-527 Biochemical compatibility, of buffers, troubleshooting experiment with 530-532 Biocompare Web site, 14 Bad data, in research, 5 Biohazards 114-117 Index 545
Index 545 glassware in, 138–139 indicator tape with, 135 microbial contamination and, 124 plastic materials in, 135 safety and, 120, 139 sterilizing membranes in, 417 time requirements for, 135 in waste decontamination, 139–140 wrapping for, 134 Autoclave settings, 133–134 Autographa californica genome, 503 Automatic temperature compensation (ATC), in pH meters, 84–85 Autoradiography film, in nucleic acid hybridization, 436–441 Avidin in hybridization buffer, 429 problems with, 395 in Western blotting, 381–382, 386–387 Axenic cultures, for protozoa, 128 Baby hamster kidney (BHK) cell lines, eukaryotic expression with, 505 BAC (bis-acrylylcystamine), as crosslinker, 338 Background noise, with storage phosphor imagers, 447 Background stain, as problem, 362, 395–396 Backup cultures, for experiments, 124 Bacteria in acrylamide polymerization, 344 disruption of, 217–218 minimizing degradation of RNA from, 215, 217–218 restriction enzymes from, 227 safe handling of, 126–127 total RNA isolation from, 208–209 Bacterial strains, maintaining, 125–126 Bacteriophages, safe handling of, 127 Baculovirus, 521–532 implementing experiment with, 527–530 insect cell line versus, 523 planning experiment with, 521–527 selecting insect cell system and, 525–527 troubleshooting experiment with, 530–532 Bad data, in research, 5 Baking membranes, 422 Balances, 51–55 calibrating, 55 factors affecting accuracy of, 51–55 in pipette testing, 72–73 selecting, 54–55 service calls for, 55 types of, 51 Banana plugs, in electrophoresis, 337 Bands after affinity purification, 485–486 fuzzy, 357 in native PAGE, 348 with pre-stained proteins, 364–365 problems with, 396 reproducible, 353 from skin keratin, 368 BandShift Kit Instruction Manual (Hennighausen & Lubon), 35 Bandwidth resolution, of spectrophotometers, 97–98 Barometric pressure, in testing pipettes, 76 Baseline flatness, of spectrophotometers, 99–100 Bases. See also Strong bases buffer absorption of, 38 buffering of, 32–33 Batch binding, in RNA purification, 211 Batch numbers, for products, 25 BCA assay, 109 Bead milling, cell disruption via, 217–218 Beckman-Coulter rotor, for centrifuges, 63, 65 Beer-Lambert law, 97, 100, 104–105, 108, 275, 277–278, 279 Bell, Peter A., 461 Below balance weighing, 53 Benzene, radioisotopes in, 147 Bequerel (Bq), 145–146n, 155 Beta emitters autoradiography film and, 438–439 shielding for, 163 Big companies, 12–13 Binding capacity, of hybridization membranes, 414 Biochemical compatibility, of buffers, 35 Biocompare Web site, 14 Biohazards, 114–117