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Detection of MIS 307 Fig. 2. The top graph shows LOH, as the peak at 104 base pairs is missing. However, the bottom graph demonstrates retention of both alleles. of the two alleles are assigned to the peaks of greatest height and the smaller peaks are interpreted as stutter bands. 3. Tumor cells are not completely separated from adjacent normal cells by microdissection. Therefore, a major advantage of the fluorescence-based method is that the loss of alleles can be determined precisely by calculation of the ratio of the peak heights of normal and tumor alleles. 4. Assays of samples with allele ratios in the border line range of 0.6 to 0.7 were repeated at least three times to ensure accuracy of results. Most repeated assays gave consistent results, and all informative markers gave similar allele ratios in the same sample. References 1. King, B. L., Carcangiu, M.-L., Carter, D., Kiechle, M., Pfisterer, J., and Kaoinski, B. M. (1995) Microsatellite instability in ovarian neoplasms. Br. J. Cancer 72, 376–382. 2. Sardi, I., Bartoletti, R., Occhini, I., Piazzini, M., Travaglini, F., Guazzelli, R., et al. (1999) Microsatellite alterations in superficial and locally advanced transitional cell carcinoma of the bladder. Oncol. Reports 6, 901–905.
308 Edwards and Bartlett 3. Sartor, O. and Sheng, A. (1997) Determinatin of CAG repeat length in the androgen receptor gene using frozen serum. Urology 49, 301–304. 4. Bronner, C. E., Baker, A. M., Morrison, P. T., Warren, G., Smith, L. G., Lesloe, M. K., et al. (1994) Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature 368, 258–261. 5. Rohrbach, H., Haas, J. C., Baretton, G. B., Hirschmann, A., Diebold, J., Behrendt, R. P., et al. (1999) Microsatellite instability and loss of heterozygosity in prostatic carcinomas: Comparison of primary tumor and of corresponding recurrences after androgen deprivation therapy and lymph node metastases. Prostate 40, 20–27. 6. Niederacher, D., Picard, F., van Roeyen, C. An, H. X., Bender, H. G., and Beckmann, W. (1997) Patterns of allelic loss on chromosome 17 in sporadic breast carcinomas dectected by fluorescent labelled microsatellite analysis. Genes Chromosomes Cancer 18, 181–192. 7. Christensen, M., Sunde, L., Bolund, L., and Orntorft, T. F. (1999) Comparison of three methods of microsatellite detection. Scand. J. Clin. Lab. Invest. 59, 167–178. 8. Serth, J., Kuczyk, M. A., Paeslack, U., Lichtinghagen, R., and Jonas, U. (2000) Quantitation of DNA extracted after microprepatation of cells from frozen and formalin-fixed tissue sections. Am. J. Pathol. 156, 1189–1196. 9. Going, J. J. and Lamb, R. F. (1996) Practical histological microdissection for PCR analysis. J. Pathol. 79, 121–124. 10. Dietmaier, W., Riedlinger, W., Kohler, A., Wegele, P., Beyser, K., Sagner, G., et al. (1999) Detection of microsatellite instability (MSI) and loss of heterozygosity (LOH) in colorectal tumours by fluorescence based multiplex microsatellite PCR. Biochemica 2, 43– 46.
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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
Page 249 and 250: 254 Ringquist et al.
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Page 269 and 270: 274 Oien 4. 100% and 70% ethanol. 5
Page 271 and 272: 276 Oien 2. Purify polyA + mRNA fro
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Page 277 and 278: 282 Oien 8. PCR. With SAGE, achievi
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Page 281 and 282: 288 Edwards and Bartlett technology
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Page 289 and 290: 296 Rithidech and Dunn 11. Ethidium
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Page 309 and 310: 316 Aubele and Smida 2.2. Chemicals
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Page 313 and 314: 320 Nakashima, Akahoshi, and Tanaka
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Page 317 and 318: 324 Stirling 9. TAE (20×): 484 g o
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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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