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T4 DNA Polymerase 471 2. Amplification conditions largely depend on the specific applications. However, a general cycling profile can be used in most experiments: 94°C for 7 min (initial denaturation); 94°C for 30 s (amplification), 55°C for 45 s, 72°C for 90 s; and 72°C for 10 min (extension). 3. Examine the PCR amplification results with agarose gel electrophoresis (see Note 1). 3.3. End Repair and Blunt-End Cloning 1. Add the following to PCR tubes directly to repair the PCR products (see Note 2): 1 U of T4 DNA polymerase; 1 UL of 4 mM dNTP solution (optional; see Note 3); 5 U of T4 polynucleotide kinase (see Note 4); and 1 µL of 1 MM ATP. 2. Incubate the reaction tubes at 25°C (room temperature) for 20 min (see Notes 5 and 6). Stop the reactions by adding 3 mL of 0.5 M EDTA, pH 8.0. 3. Incubate the reaction tubes at 70°C for 10 min to inactive the enzymes. 4. Precipitate the PCR products by adding 5 µL of 5 M NaCl and 60 µL of isopropyl alcohol (see Notes 7, ref. 8). 5. Resuspend the DNA fragments in 20 µL of TE or water. 6. Take 2 µL of DNA (containing approx 20–50 ng of PCR product) and mix with ligase and vector for ligation (see Note 8): 1 µL of 10× ligase buffer; 1 µL of T4 ligase; 2 µL of repaired DNA; dephosphorylated vector (60–150 ng); and add deionized H 2 O to a final volume of 10 µL. 7. Incubate at 16°C overnight. 8. Dilute the ligation reaction fivefold in TE buffer. Use 2 µL of the diluted ligation reaction for transformation (see Note 9). 3.4. End Repair and Sticky-End Cloning An EcoRI site is used as an example in the following protocol Fig. 1). However, depending on the desired cloning site, a different combination of dNTP should be added in the “repair” reaction (see Subheading 3.4., step 2). 1. Spin through the PCR mixture (40 µL) in a pre-equilibrated Sephacryl S-400HR spin column (see Note 10) to remove excess dNTP. 2. Add the following to the column-purified PCR fragments to generate sticky ends: 5 µL of 10× PCR buffer; 1 U of T4 DNA polymerase; 5 U of T4 polynucleotide kinase; 1 µL of 4 MM dCTP and dGTP; 1 µL of 1 MM ATP; and add deionized H 2 O to a final volume of 50 µL. 3. Incubate the reaction tubes at 25°C (room temperature) for 20 min. Stop the reaction by adding 3 µL of 0.5 M EDTA, pH 8.0. 4. Heat inactivate the enzymes by placing the reaction tubes at 70°C for 10 min. 5. Precipitate the PCR products by adding 5 µL of 5 M NaCl and 60 µL of isopropyl alcohol (8). 6. Resuspend the DNA fragments in 20 µL of TE or water. 7. Take 2 µL of DNA (containing approx 20–50 ng of DNA) and mix with ligase and EcoRI digested, dephosphorylated vector for cloning: 1 µL of 10× ligase buffer; 1 µL of T4 ligase; 2 µL of repaired DNA; dephosphorylated vector (60–150 ng); and add deionized H 2 O to a final volume of 10 µL 8. Incubate at 16°C overnight. 9. Dilute the ligation reaction fivefold in TE buffer. Use 2 µL of the diluted ligation reaction for transformation (see Note 9). 4. Notes 1. In case of multiple PCR products from a single reaction, the specific products should be purified by gel electrophoresis based on size after repair reaction. Several different
472 Wang Fig. 1. A brief outline of the strategy used to generate sticky-end PCR products with T4 DNA polymerase. methods can be used to purify DNA fragments from agarose gel, such as phenol extraction from low-melting gel (8), the “glassmilk” method, or simple low-speed centrifugation (10). The phenol extraction method has been found to be less expensive and able to recover a sufficient amount of clean DNA for cloning. 2. This protocol uses one buffer for all the enzymes that include Taq polymerase in PCR, T4 polymerase, and T4 polynucleotide kinase in end-repair reaction. Therefore, a slightly higher concentration of reagents and enzymes can be added in the reaction. 3. T4 DNA polymerase can be added directly into the PCR tube without providing additional nucleotides. However, T4 DNA polymerase balances its exonuclease and polymerase activities based on the concentration of available deoxynucleotides. Depending on the length of amplification products, number of amplification cycles, and nucleotide sequence composition of amplified region, the remaining nucleotide concentration after PCR amplification may be different from experiment to experiment. To avoid unnecessary confusion, supplemental nucleotides are routinely added for end-repair reaction. 4. T4 polynucleotide kinase is not needed when vector used has not been treated with phosphatase previously. However, dephosphorylated vector should be used to increase the cloning efficiency.
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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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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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192 Cremer and Moos Fig. 4. Testing
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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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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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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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Page 493 and 494: 506 Ravassard et al. 3.2.2. RNA Mix
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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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