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qPCR to Detect RNA Viruses 199 Fig. 1. Construction of a competitive PCR template. Figure modified from ref. 12. 2. Materials Unless stated, all chemicals are supplied by Sigma (Poole, UK), or Merck. All stock solutions up to and including the PCR step should be made using RNA free water. 2.1. Reverse Transcription (see Note 1) 1. dNTPs (1 mM; Pharmacia). 2. Anti-Sense primer (100 ng/µL). 3. RNasin (20–40 units/µL; Promega, Southampton, UK). 4. Reverse transcriptase (RT) and 10× buffer, for example, AMV (Promega). 5. Dimethyl sulphoxide (DMSO). 6. Thermocycler (Hybaid, Teddington, UK).
200 McDonagh 2.2. Basic PCR 1. dNTPs (2 mM, 10× stock; Pharmacia; see Note 2). 2. Taq or Pfu DNA polymerase and 10× buffer (Sigma, Promega; see Note 3). 3. Forward and reverse primers (2 µM; 10× stock). 4. Mineral oil (Sigma) or wax beads (Perkin–Elmer, Warrington, UK). 5. Thermocycler (Hybaid, Teddington, UK). 2.3. PCR to Create Mutated Competitor Fragments (e.g., Containing a Novel RE Site) 1. Forward and reverse primers used in conventional PCR (2 µM). 2. Internal forward and reverse primers containing bases to create novel RE site (2 µM). 3. DNA purification system (Geneclean, <strong>Bio</strong> 101, US). 4. PCR cloning system (TA cloning kit; Promega or Invitrogen; Leek, The Netherlands). 5. RE enzyme (to digest mutated product) or method of differentiating between wild-type and mutated product, such as a probe. 3. Methods 3.1. Reverse Transcription 1. Mix together the following reagents in an ice bath: 7.5 µL of RNAse free water 3 µL of DMSO 100 ng of antisense virus-specific primer (in 1 µL) 2 µL × 10 RT buffer 3 µL of 1 mM dNTPs 0.5 µL of RNAsin (20–40 units/µL) 5 µL of RNA, prepared by extraction method 1 µL of RT 2. Incubate for 30–60 min at 42°C followed by 5 min at 95°C and 5 min at 4°C. 3. Pulse spin tubes and add 5 µL to PCR reaction or store at –20°C until required (see Note 4). 3.2. PCR 3.2.1. Basic Reaction 1. Make up a master mix containing 5 µL of buffer (×10); 5 µL of dNTP (2 mM, final concentration 0.2 mM); 5 µL of each primer (2 µM, final concentration 0.2 µM); and 1 U DNA polymerase (see Notes 2 and 3). 2. Overlay with oil if not using a thermocycler with a heated lid, then add 5 µL of prepared cDNA through the oil. 3. A typical cycling protocol consists of denaturation at 95°C for 30 s, annealing at 5°C below the primer melting point for 30 s, and extension at 68 to 74°C for 45 s (see Note 5). 4. Carry out as few cycles as necessary because the reaction is only linear over a short range and this may distort the results. 5. To further increase sensitivity, it is beneficial to use nested PCR. As a general rule, the primary round should consist of 20 to 25 cycles and the secondary round up to 20 cycles. It may also be necessary to add diluted primary product (1/10) to the secondary round. 3.2.2. Limiting Dilution Method A limiting dilution of cDNA is performed in 10-fold steps, and this is then added to the PCR. The sensitivity of the assay must be known, and the amount of cDNA in
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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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144 Benjamin, Smith, and Waites 3.3
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Page 147 and 148: 148 Benjamin, Smith, and Waites 8.
Page 149 and 150: 150 Benjamin, Smith, and Waites
Page 151 and 152: 152 Olmos et al. Fig. 1. RT-hemines
Page 153 and 154: 154 Olmos et al. This nested RT-PCR
Page 155 and 156: 156 Olmos et al. Table 2 Volume and
Page 157 and 158: 158 Olmos et al. 8. The sensitivity
Page 159 and 160: 160 Olmos et al.
Page 161 and 162: 162 Abe 5. Moloney murine leukemia
Page 163 and 164: 164 Abe Table 2 Detection Rate of H
Page 165 and 166: 166 Abe 4. For HBV, nested PCR usin
Page 167 and 168: 168 Tellier et al. of Pfu (and ther
Page 169 and 170: 170 Tellier et al. 2. Cline, J., Br
Page 171 and 172: 172 Tellier et al. 38. Tamiya, S.,
Page 173 and 174: 174 Tellier et al. 13. Thin-wall PC
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Page 179 and 180: 182 Stirling efficiency of the reac
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Page 183 and 184: 186 Cremer and Moos Fig. 1. Rearran
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Page 195: 198 McDonagh the dilution method is
Page 199 and 200: 202 McDonagh Fig. 2. Quantitative P
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Page 203 and 204: 206 Bartlett Table 1 Example Assay
Page 205 and 206: 208 Bartlett 3.4. Calculation of Re
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Page 211 and 212: 214 Kerr after the PCR. This reduce
Page 213 and 214: 218 Bartlett As with any experiment
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Page 221 and 222: 226 Dominguez et al. Table 1 Primer
Page 223 and 224: 228 Dominguez et al. 4. Oligonucleo
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Page 227 and 228: 232 Dominguez et al. 3.4. Gel Elect
Page 229 and 230: 234 Dominguez et al. 3. If the cont
Page 231 and 232: 236 Dominguez et al. 11. Joshi, C.
Page 233 and 234: 238 Khalturin, Kuznetsov, and Bosch
Page 241 and 242: 246 Ringquist et al. In this chapte
Page 243 and 244: 248 Ringquist et al. 5. Total RNA s
Page 245 and 246: 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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406 Speel, Ramaekers, and Hopman Ta
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408 Speel, Ramaekers, and Hopman Fi
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410 Speel, Ramaekers, and Hopman po
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Table 2 Comparison of PRINS, in Sit
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414 Speel, Ramaekers, and Hopman te
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Table 3 Summary of Control Experime
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418 Speel, Ramaekers, and Hopman 3.
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420 Speel, Ramaekers, and Hopman ad
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422 Speel, Ramaekers, and Hopman 25
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426 Bull and Paskins Fig. 1. Result
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428 Bull and Paskins 2.3. Detection
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430 Bull and Paskins 6. Shake the s
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432 Bull and Paskins
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434 Wiedorn and Goldmann Fig. 1. IS
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436 Wiedorn and Goldmann 41. Protei
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438 Wiedorn and Goldmann 2. Incubat
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440 Wiedorn and Goldmann 3.15. Prim
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442 Wiedorn and Goldmann ficient se
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444 Wiedorn and Goldmann 25. Kommin
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446 Gilchrist and Befus Fig. 1. Sch
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448 Gilchrist and Befus 2. Cells ar
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450 Gilchrist and Befus Fig. 3. RT
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452 Gilchrist and Befus References
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454 Speel, Ramaekers, and Hopman of
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456 Speel, Ramaekers, and Hopman Fi
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462 Speel, Ramaekers, and Hopman bu
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468 Pearson and Stirling into PCR p
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470 Wang 2. Materials 2.1. PCR 1. D
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472 Wang Fig. 1. A brief outline of
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474 Wang
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476 Horton, Raju, and Conti-Fine Fi
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478 Horton, Raju, and Conti-Fine th
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480 Horton, Raju, and Conti-Fine 1.
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482 Horton, Raju, and Conti-Fine ot
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484 Horton, Raju, and Conti-Fine
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486 Preston amplification approach
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488 Preston 1. 10× PCR buffer: 100
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490 Preston Fig. 1. Design of degen
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492 Preston 2. Remove the AmpliTaq
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494 Preston 3.3. Cloning and DNA Se
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496 Preston MgCl 2 concentrations b
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498 Preston 21. Hung, T., Mak, K.,
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500 Ravassard et al. Fig. 1. Constr
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502 Ravassard et al. size dispersio
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504 Ravassard et al. 9. ddH 2 O. 10
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506 Ravassard et al. 3.2.2. RNA Mix
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508 Ravassard et al. 4. Wash twice
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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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