Appendix

Materials, Equipment, and Reagents

• Materials

- O.5-ml regular PCR Eppendorf (Eppendorf) - O.S-ml thin-walled PCR Eppendorf tubes (Eppendorf) - loS-ml Eppendorf tubes (Eppendorf) - 3MM filter paper (Whatman) - IS-ml Corex tubes - SO-ml disposable tubes (Falcon, BD) - Aerosol-resistant tips (ART, BMP) - Disposable lO-ml pipettes (Falcon, BD) - Disposable SO-ml tubes (Falcon, BD) - Disposable gloves (BD) - Disposable pipettes (Falcon, BD) - Dry ice - Electroporation apparatus (Invitrogen) - Electroporation cuvettes (Invitrogen) - Liquid nitrogen (N2)

- Mini-microtiter plates (Nalgene) - Nylon filters (Hybond N+, Amersham) - Oak-Ridge tube (Nalgene, SO-ml polyallomer) - Pasteur pipettes - Sterile glass beads - Sterilized scissors - Yellow and blue tips (Gilson) - Adjustable hand pipettors (Gilson)

• Equipment

- Automatic pipette-aid (Falcon, BD) - Geiger ~-counter (e.g., Camberra) - Centrifuge (e.g., Beckman) - Electrophoretic apparatus (e.g., Stratagene)

Materials, Equipment, and Reagents III

- Electroporator apparatus (Invitrogen) - Fume hood (e.g., ICN) - High-voltage power supply (e.g., Pharmacia) - Microfuge (e.g., Eppendorf) - Mortar and pestle - PCR thermocycler (e.g., Perkin-Elmer) - Quartz cuvettes - Sequencing apparatus (e.g., Stratagene) - Slot blot apparatus (Schleicher & Schuell) - Spectrophotometer (e.g., Beckman) - Thermostated waterbath (e.g., New Brunswick) - Timer - Tissue homogenizer (e.g., Polytron, Dounce) - UV transilluminator (e.g., Fotodyne) - Vacuum dryer (e.g., Bio-Rad) - Vacuum dryer centrifuge (e.g., Savant) - Vacuum pump (e.g., Edwards) - Vortex (e.g., Heidolph)

• Reagents

- 8-Hydroxyquinoline (J.T. Baker) - 30 % H20 2 (J.T. Baker) - 75 % ethanol in DEPC-2dH20 - 80 % ethanol in DEPC-2dH20 - 98 % ethanol (J.T. Baker) - Acrylamide (Bio-Rad) - Agarose (Sigma) - Ammonium acetate (NH40AC, J.T. Baker) - Ammonium persulfate (APS, Bio-Rad) - Ampicillin (Sigma) - Ampli Taq (Perkin-Elmer) - ATP, GTP, CTP, UTP (Gibco-BRL) - ~-Mercaptoethanol (J.T. Baker) - Bacto-agar (Oxoid) - Bacto-tryptone (Oxoid) - Bacto-yeast extract (Oxoid) - bis-Acrylamide (Bio-Rad) - Boric acid (J.T. Baker) - Bromophenol blue (Bio-Rad) - Calcium chloride (CaC12, J.T. Baker) - Calf intestinal phosphatase enzyme (CIP, Boehringer)

112 ApPENDIX

- Chloroform (CHCh, J.T. Baker) - CIP buffer lOx (Boehringer) - dATP, dCTP, dGTP, dTTP (dNTPs, Gibco-BRL) - Deionized formamide (Gibco-BRL) - DEPC-2dH20 - Diethyl-pyrocarbonate (DEPC, Sigma) - Dimethylsulfoxide (DMSO Fluka) - Dithiotreitol (DTT, Sigma) - DNA marker V (Boehringer) - DNAse I (GenHunter) - Ethidium bromide (EtBr, Sigma) - Ethylendiamine-tetraacetic acid (EDTA, Sigma) - Formaldehyde (Fluka) - Formamide (Gibco-BRL) - Glacial acetic acid (J.T. Baker) - Glucose (Sigma) - Glycerol (Sigma) - Glycogen (GenHunter) - Guanidine thiocyanate (Sigma) - H-AP random primers - HEPES (Sigma) - Isoamyl alcohol (J.T. Baker) - Isopropanol anhydrous (J.T. Baker) - Left primer (sequence depends on the vector used) - Lithium chloride (LiCtz, Sigma) - M-MLV reverse transcriptase (Gibco-BRL) - Magnesium chloride (MgC12, Sigma) - Megaprime DNA labeling system (Amersham, RPN160617) - Message Clean Kit (GenHunter) - MgC12 (Perkin-Elmer) - Mineral oil (Chill-out liquid wax, MJ Research) - Morpholinpropane sulfonic acid (MOPS, Sigma) - N-Lauryl-sarkosine (Sarkosyl, Sigma) - Na citrate (J.T. Baker) - Nonidet P-40 (NP-40, Sigma) - Nucleic acid-grade agarose (Sigma) - One-base-anchored oligo H-TuA - One-base-anchored oligo H-Tue - One-base-anchored oligo H-TuG - PCR buffer (Perkin-Elmer) - Phenol (nucleic acid grade, Genenco) - PIPES (Sigma) - PK buffer lOx (Boehringer)

Materials, Equipment, and Reagents 113

- Potassium acetate (KOAc, J.T. Baker) - Potassium chloride (KCI, J.T. Baker) - Proteinase K (Sigma) - Reverse transcription buffer (Gibco-BRL) - Right primer (sequence depends on the vector used) - RNase A (Sigma) - RNase TI (Boehringer) - RNase ONE (Promega) - RNasin (Promega) - Sequenase Kit (USB) - Sigmacote (Sigma) - Sodium acetate (NaOAc, J.T. Baker) - Sodium chloride (NaCI, J.T. Baker) - Sodium dihydrogen phosphate ((NaH2P04, J.T. Baker) - Sodium dodecyl sulfate (SDS, Sigma) - Sodium hydrogen phosphate (Na2HP04, J.T. Baker) - Spermidine (Sigma) - t-RNA (Sigma) - T4 polynucleotide kinase (PK, 1 U/fA-I, Boehringer) - T7, T3, or SP6 RNA polymerase (20 U/fA-1 Promega) - TA cloning kit (Invitrogen) - TEMED (Bio-Rad) - Thermo-Sequenase cycle sequencing kit (Amersham) - Trizma base (Sigma) - Tween 20 (Sigma) - Urea (Sigma) - X-gal (Sigma) - Xylene cyanol FF (Bio-Rad) - [a32PjdCTP (3000 Ci/mmol, Amersham) - [a32PjUTP (800Ci/mmol, Amersham) - [a35SjdATP (1200 Ci/mmol, Amersham) - [y32p jATP (3000 Ci/mmol, Amersham)

• Suppliers

Amersham International plc Amersham Place, Little Chalfont Buckinghamshire, UK

BD, Becton Dickinson Labware 2 Bridgewater Lane Lincoln Park, New Jersey, USA

114 ApPENDIX

Beckman Stanford Industrial Park Palo Alto, California, USA

Bio-Rad Laboratories 2000 Alfred Nobel Drive Hercules, California, USA

Boehringer Mannheim GmbH Sandhofer Strasse 116 Mannheim, Germany

Braun Melsungen AG Postfach 11 0 D-3508 Melsungen, Germany

Camberra Industries Inc. One State Street 06450 Meriden, Connecticut, USA

Eppendorf Netheler Hins GmbH Bakhausenweg 1 Hamburg, Germany

Fluka Chemika Biochemika Fluka Chemie AG Industriestrasse 25 Buchs, Switzerland

Fotodyne Inc. 16700 West Victor Road 9980 New Berlin, Wisconsin, USA

GenHunter Corporation 50 Boylson Street Brookline, Massachusetts, USA

Gibco-BRL Life Techonologies Ltd Trident House, Renfrew Road PA34EF Paisley, UK

Gilson Medical Electronics S.A. Rue Gambetta 72 95400 Villiers Le Bel, France

Invitrogen Corp. 39858 Sorrento Walley Blvd San Diego, California, USA

J.T. Baker B.V. Rijstenborgherweg 20 Deventer, The Netherlands

MBP, Molecular BioI-Products Waples Street 9888 San Diego, California, USA

Nalgene Ltd. Rotherwas HR2 6JQ Hereford, UK

Materials, Equipment, and Reagents 115

New Brunswick Scientific Co. Inc. Talmadge Road 44 08818 Edison, New Jersey, USA

Oxoid Ltd. Wade Road Basingstoke RG24 OPW Hampshire, UK

Perkin-Elmer Corp. 850 Lincoln Centre Drive Foster City, California, USA

Pharmacia Biotech Pharmacia Biosystems AB 75183 Upsala, Sweden

Prom ega Corp. 2800 Woods Hollow Road Madison, WI, USA

Schleicher & Schuell GmbH Hahnestrasse 3 3354 Dassel, Germany

Sigma Chemical Company 3050 Sprouce Street 63178 St. Louis, Missouri, USA

Stratagene 11011 North Torrey Pines Road 92037 La Jolla, California, USA

Whatman Ltd. Springfield Mill Maidstone ME 1421 Kent, UK

USB, United States Biochemical Corp. P.O. Box 22400 44122 Cleveland, Ohio, USA

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Subject Index

acrylamide 49 affmity capturing 81, 96, 101 agarose - denaturing agarose gels 16 - gel 29 - low-melting agarose 64, 68 - mini-gel 70 - ampicillin 75 annealing - of primers 40 - temperature 10,40,59 antisense RNA probes 88

bacterial culture 73

cDNA - cloning of amplified cDNA frag­

ments 64 - differential expression of cDNA

fragments 81 - duplex 39 - elution of cDNA fragments 57 - purification of cDNA fragments

from agarose 68 - reamplification of eluted

cDNAs 59 - separation of cDNA fragments

46 - synthesis 36 - template 45 checking subcloned fragments 77 ClP dephosphorylation buffer 53 colony lysis buffer 79

DNA - chromosomal DNA contami­

nation 29 - genomic DNA contamination

29

- labeling 85 - loading dye 49 - markers 51 - polymerase used for differential

display 40 - recovery of 68

-- sequencing 102 - thermocycler 108 DNase treatment 29

E. coli 64, 71 electro competent cells 73 electroporation 76 ethidium bromide 32 - staining 16,29,46 - alignment of gel image fIlm 55

fluorescence labeling 41

guanidine - thiocyanate 15,21,22,24 - thiocyanate extractions 16

hybridization 85 hybridization buffer 90

in vitro transcription 88 insert - amplification 95 - checking 77 - checking buffer 79 - purification 64

Klenow fragment 86

labeling - [a32PjdATP 41 - [a32PjdCTP 41 - [a32PjdNTPs 41

- [a33PldATP 41 - [a35S1dATP 41 - end labeling of DNA markers

51 - random 85 lacZa 64 LB - plates 64, 74 - broth 72 ligase - T4 DNA 71 ligation - blunt -end 64 - efficiency 64, 70 - of cDNAs 70 - of PCR inserts 64 lysis buffer 93

M-MLV reverse transcriptase 73 master amplification mixes 42, 98 MOPS buffer 83 multiple probes 88

northern blot 83, 84 nuclear - freezing buffer 93 - run-on 81,92 - transcription 94

oligo - anchored oligo 37, 38,42, 59,

99 - base-anchored 98

PCR - amplification 36, 39 - labeling 98 - thermocycler 36, 59, 77, 98 pCR-TRAP 64 - cloning 64 pCR2.1 64 phosphate buffer 86 PK buffer 53 plasmid - DNA 105 polyacrylamide gel - denaturing 46 - nondenaturing 46 prehydridization and hybridization

mix 86

Subject Index 123

primer - anchored oligo-dT primers 9 - annealing 39 - arbitrary 11 - downstream 34 - design of 3-end primers 9 - design of 5'-end primers 10 - design of primers 40 d-TlzMN primers 9 - independent primers 12 - number and length of primers

11 - upstream 12 probe-target 88 radioisotope - choice of 41 random annealing 11 ribonuclease protection assay 88 RNA - concentration 29 - degradation 15 - extraction

by guanidine thiocyanate 21 from fish liver 25 from animal cells 24 from plant tissues 21 from yeast 18

integrity of total RNA 31 - long storage 23 - phenol/CHCl3 buffer 18 - poly-A+ RNA 16 - preparation of labeled RNA 94 - preparation of total RNA 15 - probe 88, 90, 95 - ribosomal RNAs 16 - quantification 28 - visualization by UV 32 RNase 20 - contaminant 84 - contamination 31 - DNase-free RNase A 104 - digestion mixture 90 - inactivation of endogenous

RNase 15 - inhibitors 15 - protection assay 81, 88 - stop mix 90 RNase-free DNase I 29 RNasin 20 rUTP labeling mix 89

124 Subject Index

Sequenase version 2.0 106 Sequencing lO2, 106 slot blot 83 SOC medium 74 SSPE 93 STE 93 sub cloned fragments 77 T7 DNA polymerase 106 T7, T3, SP6 polymerase 88,89 TA cloning 64 Taq polymerase 40

transcription buffer 89, 93 transformation - of CaCl2 competent cells 74 - of competent cells 71 - of competent cells by electro-

poration 75 trituration buffer 72

UV-fIx 84

vector 64, 71