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PRINS and Immunocytochemistry 457 9. Washing buffer: 4 × SSC (diluted from 20 × SSC, see Subheading 2.1.2., item 15), 0.05% Triton X 100. 10. Blocking buffer: 4 × SSC (diluted from 20 × SSC, see Subheading 2.1.2., item 15), 0.05% Triton X 100, 5% dried skimmed milk powder. 11. Glass slides (Menzel). 12. Coplin jars. 13. Cytocentrifuge (Shandon, Astmoor, UK). 14. Waterbath at 70°C. 15. Thermal cycler (Hybaid Omnigene Flatbed; Hybaid). 16. Incubator at 37°C. 2.2.2. Immunofluorescence Detection of Mitotic Spindle 1. Mouse monoclonal anti-β-tubulin antibody (Sigma). 2. TRITC-conjugated donkey anti-mouse IgG F(ab′)2 fragments (DAMTRITC; Jackson Immunoresearch, West Grove, PE). 3. Normal goat serum (NGS). 4. Vectashield (Vector). 5. DAPI (see Subheading 2.1.2., item 11). 6. TOTO-3 iodide (Molecular Probes, Eugene, OR). 7. Blocking buffer: 1× PBS (diluted from stock 10× PBS, see Subheading 2.1.1., item 9), 0.05% Triton X-100, 2-5% NGS. 8. Washing buffer: 1× PBS (diluted from stock 10× PBS, see Subheading 2.1.1., item 9), 0.05% Triton X-100. 9. 1× PBS. 10. Vectashield (Vector) containing 0.5 µg/mL DAPI or 3000× diluted TOTO-3 iodide (from 1 mM stock in DMSO). 11. Humid chamber. 12. Coplin jar (50 or 100 mL). 13. Fluorescence microscope, CCD camera, and confocal microscope (see Subheading 2.1.2., items 27–30). 2.3. Protocol 3 2.3.1. Immunofluorescence Detection of Structural Proteins in Chromosomes 1. Normal goat serum (NGS). 2. Primary antibody. 3. FITC-conjugated secondary antibody. 4. Alcohol-cleaned glass slides and coverslips (Menzel). 5. Hypotonic solution: 75 mM KCl. 6. Potassium chromosome medium (KCM) solution: 120 mM KCl, 20 mM NaCl, 10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, and 0.1% (v/v) Triton X-100. 7. Blocking buffer: KCM containing 2 to 5% NGS. 8. Fixation solution: KCM containing 10% formalin (from 37% stock solution; Merck). 9. Cytocentrifuge (Shandon). 10. Coplin jar (50 or 100 mL). 11. Humid chamber. 2.3.2. PRINS DNA Labeling 1. Immersion solution: 0.1 M NaOH. 2. Neutralization solution: 0.01 M Tris-HCl, pH 7.4.
458 Speel, Ramaekers, and Hopman 3. Methanol:acetic acid (31). 4. A series of cold (4°C) 70, 96, and 100% ethanol. 5. Denaturation solution: 30 mM NaOH/1 M NaCl (pH >12). 6. PRINS reaction, detection, and mounting components, as described in Subheading 2.1.2., items 2–26. 7. Slide evaluation equipment, as described in Subheading 2.1.2., items 27–30. 3. Methods 3.1. Protocol 1 3.1.1. Fluorescence Immunophenotyping by Alkaline Phosphatase Cytochemistry 1. Hybrid (C121-TN6, J1-C14) and tumor (H460) cell lines are cultured on glass slides by standard methods (20,22,23), fixed in either cold methanol (–20°C) for 5 s and cold acetone (4°C) for 3 × 5 s, cold acetone (–20°C) for 10 min, or 70% ethanol (–20°C) for 10 min, air-dried, and stored at –20°C until use (see Note 3). 2. Incubate slides for 10 min at room temperature with 100 µL of blocking buffer under a coverslip in a humid chamber. 3. Remove the coverslip, discard the blocking buffer, and incubate the slides for 30 to 45 min at room temperature with 50 µL of undiluted culture supernatant of the appropriate antigenspecific monoclonal antibody containing 2% NGS under a coverslip in a humid chamber. 4. Wash slides for 2 × 5 min with washing buffer in a Coplin jar. 5. Incubate slides for 30 to 45 min at room temperature with 50 µL of GAMAPase, diluted 150 in blocking buffer (see Note 4), under a coverslip in a humid chamber. 6. Wash slides for 5 min with washing buffer, and for 5 min with 1× PBS. 7. Visualize the antigen with the alkaline phosphatase-Fast Red (APase-Fast Red) reaction: Mix 4 mL of APase buffer, 1 mg of naphthol-ASMX-phosphate in 250 µL of buffer without PVA and 5 mg of Fast Red TR in 750 µL of buffer without PVA just before use and overlay each sample with 100 µL under a coverslip. Incubate the slides for 5 to 15 min at 37°C and wash 3× 5 min with 1× PBS (see Notes 5–7). 3.1.2 PRINS DNA Labeling 1. Process cells for PRINS as follows: wash slides for 2 min at 37°C with 0.01 M HCl, incubate the samples with 100 µg/mLpepsin in 0.01 M HCl for 20 min at 37°C, wash again with 0.01 M HCl for 2 min, and post-fix the slides in 1% formaldehyde in 1× PBS for 10 min at room temperature. Wash cells in 1× PBS for 5 min at room temperature, followed by a wash step in 1× Taq buffer for 5 min at room temperature (all steps in a Coplin jar). 2. Prepare the PRINS reaction mix on ice as follows: Dilute 100 mM dATP, dGTP, and dCTP 110 with distilled water. Dilute 100 mM dTTP 1:100. Put together in a microcentriphuge tube: 1 µL of each of the diluted dNTPs, 1 µL of either 1mM <strong>Bio</strong>tin-16-dUTP, Digoxigenin- 11-dUTP, or Fluorescein-12-dUTP (see Note 8), 5 µL of 10× Taq buffer, 250 ng of oligonucleotide (see Note 9), 1 U Taq polymerase, and distilled water to 50 µL. 3. Place 40 µL of this mixture under a coverslip on the slide, seal with rubber cement, air-dry the rubber cement, and transfer to the heating block of the thermal cycler. 4. Each PRINS reaction cycle consists of 2 min at 94°C (denaturation of cellular DNA, see Note 10), 5 min at the appropriate annealing temperature (see Note 11) and 15 min at 72°C for in situ primer extension. 5. Stop the PRINS reaction by transferring the slides (after removal of the rubber solution seal) to 50 mLof PRINS stop buffer in a Coplin jar at 65°C for 1 min (see Note 12). 6. Transfer the slides to washing buffer at room temperature and wash 5 min.
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TM Methods in Molecular Biology Vol
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Xin Wang and W. Scott Young III 105
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63. Primed In Situ Nucleic Acid Lab
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4 Bartlett and Stirling The thing t
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6 Bartlett and Stirling Fig. 2. Res
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8 Carroll and Casimir of PCR. Such
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10 Carroll and Casimir cost more th
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12 Carroll and Casimir are no longe
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14 Carroll and Casimir Should these
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16 McDonagh Fig. 1. Unidirectional
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18 McDonagh stored. This means that
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20 McDonagh
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22 Stirling which will either not a
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24 Stirling
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28 Bartlett References 1. US Depart
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30 Bartlett and White 11. 5 M sodiu
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32 Bartlett and White
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34 Pearson and Stirling 11. Microfu
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36 Going 3. Methods 3.1. Section Cu
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38 Going pipet filler. Spread the p
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40 Going digitally to record the di
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42 Going
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44 Pearson 7. Add 60 µL of chlorof
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46 Bartlett 3. Methods 3.1. RNA Ext
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48 Pearson 3. Method The method of
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50 Stirling and Bartlett 4. Protein
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52 Stirling and Bartlett
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54 Stirling 3. Methods 3.1. Fungal
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56 McDonagh 2. Add 0.4 mL of TNE bu
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58 Schmerer 2. Materials To apply t
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60 Schmerer 3. The additional purif
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62 Schmerer
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64 Stirling
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66 Bartlett Table 1 Recommended Aga
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68 Bartlett Table 2 Separation of D
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70 Bartlett 2. 5× TBE: 54 g of Tri
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72 Bartlett 7. Remove upper aqueous
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74 Bartlett 4. Many modern electrop
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76 Bartlett
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78 Stirling 11. Store at -20°C for
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82 Hyndman and Mitsuhashi 3.1. Effi
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84 Hyndman and Mitsuhashi Fig. 1. H
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86 Hyndman and Mitsuhashi Fig. 3. H
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88 Hyndman and Mitsuhashi can be ge
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90 Grunenwald may be affected by ea
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92 Grunenwald 50°C will generally
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94 Grunenwald of template DNA, a su
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96 Grunenwald than absolutely neces
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98 Grunenwald 2. Foord, O. S. and R
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100 Grunenwald
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102 Stirling Table 1 Thermal Cycler
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104 Stirling
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106 Wang and Young full-length cDNA
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108 Wang and Young Fig. 1. (A) Nort
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110 Wang and Young Fig. 3. Expresse
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112 Wang and Young 2. First-strand
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114 Wang and Young 3′-RACE outlin
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116 Wang and Young
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118 Dassanayake and Samaranayake co
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120 Dassanayake and Samaranayake 5.
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122 Dassanayake and Samaranayake Re
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124 Iannone et al. Fig. 1. Diagram
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Table 1 Oligonucleotides Descriptio
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128 Iannone et al. 5. For microsphe
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130 Iannone et al. 4. To adjust for
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132 Iannone et al. 6. Target concen
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134 Iannone et al.
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136 Benjamin, Smith, and Waites amp
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138 Benjamin, Smith, and Waites the
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140 Benjamin, Smith, and Waites 2.2
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142 Benjamin, Smith, and Waites 2.
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148 Benjamin, Smith, and Waites 8.
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150 Benjamin, Smith, and Waites
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152 Olmos et al. Fig. 1. RT-hemines
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154 Olmos et al. This nested RT-PCR
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156 Olmos et al. Table 2 Volume and
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158 Olmos et al. 8. The sensitivity
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160 Olmos et al.
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162 Abe 5. Moloney murine leukemia
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164 Abe Table 2 Detection Rate of H
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166 Abe 4. For HBV, nested PCR usin
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168 Tellier et al. of Pfu (and ther
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170 Tellier et al. 2. Cline, J., Br
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172 Tellier et al. 38. Tamiya, S.,
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174 Tellier et al. 13. Thin-wall PC
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176 Tellier et al. 13 min for the l
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178 Tellier et al.
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182 Stirling efficiency of the reac
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184 Stirling
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186 Cremer and Moos Fig. 1. Rearran
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188 Cremer and Moos 4. Count cells
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190 Cremer and Moos Fig. 3. Alignme
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192 Cremer and Moos Fig. 4. Testing
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194 Cremer and Moos 5. Compare the
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196 Cremer and Moos 11. Klein, E.,
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198 McDonagh the dilution method is
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200 McDonagh 2.2. Basic PCR 1. dNTP
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202 McDonagh Fig. 2. Quantitative P
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204 McDonagh
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206 Bartlett Table 1 Example Assay
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208 Bartlett 3.4. Calculation of Re
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210 Bartlett 11. Cerenkov counting
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212 Kerr Fig. 1. Fluorescent probes
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214 Kerr after the PCR. This reduce
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218 Bartlett As with any experiment
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220 Bartlett Even once novel regula
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222 Bartlett Notwithstanding these
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224 Bartlett
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226 Dominguez et al. Table 1 Primer
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228 Dominguez et al. 4. Oligonucleo
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230 Dominguez et al. Fig. 2. cDNA n
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232 Dominguez et al. 3.4. Gel Elect
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234 Dominguez et al. 3. If the cont
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236 Dominguez et al. 11. Joshi, C.
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238 Khalturin, Kuznetsov, and Bosch
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246 Ringquist et al. In this chapte
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248 Ringquist et al. 5. Total RNA s
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250 Ringquist et al. 3. Purificatio
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252 Ringquist et al. 2. The present
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254 Ringquist et al.
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256 Case-Green, Pritchard, and Sout
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272 Oien Fig. 1. A schematic diagra
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274 Oien 4. 100% and 70% ethanol. 5
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276 Oien 2. Purify polyA + mRNA fro
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278 Oien 50-mL conical tubes. Resus
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280 Oien Forward Primer, and then r
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282 Oien 8. PCR. With SAGE, achievi
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284 Oien
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288 Edwards and Bartlett technology
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290 Edwards and Bartlett ing less p
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292 Edwards and Bartlett Stepwise m
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294 Edwards and Bartlett
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296 Rithidech and Dunn 11. Ethidium
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298 Rithidech and Dunn Fig. 1. PCR
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300 Rithidech and Dunn
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302 Edwards and Bartlett Studies th
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304 Edwards and Bartlett 11. Hot st
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306 Edwards and Bartlett Fig. 1. Ex
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308 Edwards and Bartlett 3. Sartor,
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310 Schmerer the investigation conc
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312 Schmerer References 1. Kunkel,
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314 Schmerer
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316 Aubele and Smida 2.2. Chemicals
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318 Aubele and Smida References 1.
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320 Nakashima, Akahoshi, and Tanaka
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322 Nakashima, Akahoshi, and Tanaka
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324 Stirling 9. TAE (20×): 484 g o
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326 Stirling
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328 Han and Robinson Fig. 1. Basic
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330 Han and Robinson sequence diffe
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332 Han and Robinson 4. Notes 1. PC
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334 Han and Robinson
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338 Stirling of simple and improved
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340 Stirling concentrations interfe
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342 Daniels to sequence a 500-bp PC
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344 Daniels 4. Load all of the samp
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346 Daniels References 1. Orita, M.
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348 Kösel et al. Fig. 1. Schematic
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350 Kösel et al. 3. Methods 3.1. P
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352 Kösel et al. Fig. 2. Sequencin
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354 Kösel et al. 5. Kwok, S. and H
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356 Mazars and Theillet Fig. 1. Sch
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358 Mazars and Theillet of genomic
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360 Mazars and Theillet
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362 Suomalainen and Syvänen Fig. 1
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364 Suomalainen and Syvänen detect
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366 Suomalainen and Syvänen temper
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368 Shen et al. ladder. Also, direc
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370 Shen et al. 3. Mineral oil. 4.
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372 Shen et al. extended primers, w
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374 Quivy and Becker Fig. 1. The us
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376 Quivy and Becker that decreases
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378 Quivy and Becker 2. Solution of
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380 Quivy and Becker 7. Precipitate
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382 Quivy and Becker 7. Prepare a p
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384 Quivy and Becker
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386 Walsh by most, but not all M13
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388 Walsh PCR products are now flus
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390 Walsh 5. For palindromic restri
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392 Walsh
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394 McAleer, Coffey, and Dunham Fig
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396 McAleer, Coffey, and Dunham Tab
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398 McAleer, Coffey, and Dunham Tab
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400 McAleer, Coffey, and Dunham
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402 Stirling 5. Homopolymer Regions
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Page 461 and 462: 474 Wang
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Page 473 and 474: 486 Preston amplification approach
Page 475 and 476: 488 Preston 1. 10× PCR buffer: 100
Page 477 and 478: 490 Preston Fig. 1. Design of degen
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Page 481 and 482: 494 Preston 3.3. Cloning and DNA Se
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Page 485 and 486: 498 Preston 21. Hung, T., Mak, K.,
Page 487 and 488: 500 Ravassard et al. Fig. 1. Constr
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Page 491 and 492: 504 Ravassard et al. 9. ddH 2 O. 10
Page 493 and 494: 506 Ravassard et al. 3.2.2. RNA Mix
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510 Ravassard et al.
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512 Pont-Kingdon Fig. 1. Constructi
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514 Pont-Kingdon 9. Invert tube and
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516 Pont-Kingdon
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518 Jones and Winistorfer Fig. 1. D
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520 Jones and Winistorfer Fig. 2. D
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522 Jones and Winistorfer The yield
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524 Jones and Winistorfer 7. Goulde
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526 Burke and Barik Fig. 1. The bas
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528 Burke and Barik concentration o
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530 Burke and Barik purified mutant
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532 Burke and Barik
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