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Direct and Indirect In Situ PCR 441 of 200 to 800 bp because with larger amplicons the amplification efficiency will be reduced dramatically and will often result in false negative samples. 5. Because dextran sulfate may produce background staining, especially if higher concentrations are used, reduce the amount of dextran sulfate to diminish the background staining. For ISH in contrast to filter hybridization, usually 10% dextran sulfate is the upper limit. 6. The reverse transcription can also be performed with other transcriptases, such as Superscript (GibcoBRL, Karlsruhe, Germany). In this case, the protocol has to be adjusted accordingly. 7. For SuperFrostPlus slides, this is only a precaution as when stored (dust free) and handled under appropriate conditions, we have encountered neither DNAse nor RNAse contamination. 8. Coverslips may be siliconized prior to the incubation at 220°C if adherence of tissue sample to the coverslips is observed during the PCR procedure. 9. To obtain reproducible results, it is essential that fixation is always performed at the same temperature and for the same time. Otherwise, the permeabilization conditions have to be adapted and optimized for each PCR. A difference of 5°C during fixation may give false negative results because of changes in cell permeability. Unfortunately, tissue in molecular pathology is usually not fixed under standardized conditions. Therefore, several permeabilization conditions have to be tested for each sample. To improve fixation and therefore DNA and RNA preservation, tissues should be cut to the minimum size prior to fixation. For mRNA detection paraformaldehyde will yield a better preservation of RNA than formalin. Perform fixation at 4°C where possible. The extraction of RNA from formalinor paraformaldehyde-fixed tissues for doing a solution-phase PCR to verify the in situ results especially when establishing a new protocol is usually very ineffective. For these applications, we recommend the use of the Hope fixative (patent pending, Dr. Olert, Institut für Kinderpathologie, Universität Mainz, Germany available at DCS, Hamburg, Germany) which additionally yields a much better preservation of RNA compared to other fixatives. 10. Thinner sections show better adhesion to slides during IS-PCR, although this reduces the number of target sequences, which may result in some negative cells. 11. Use fresh reagents for each incubation. Because xylenes saturate rapidly with paraffin use 200 mL of fresh xylene for each batch of 15 to 20 slides. 12. Although fixed under the same conditions, different tissues may require adapted permeabilization parameters. When optimizing permeabilization parameters with respect to morphology, it is usually better to prolong Proteinase K incubation than to increase the concentration of Proteinase K (15). If diffusion artifacts are a major problem the reduction of the concentration of Proteinase K with simultaneous prolongation of incubation turned out to be advantageous (15). If you encounter background staining this may be reduced by the use of target retrieval instead of Proteinase K (15). Proteinase K (250 µg/ mL) is optimal for portio and condylomata biopsies fixed for 24 h in buffered formalin whereas for leukocytes 10 to 30 µg/mL Proteinase K and for bronchial epithelial cells 30 to 50 µg/mL Proteinase K performed well. Leukocytes and bronchial epithelial cells were fixed with 4% buffered paraformaldehyde for 30 min. 13. The concentration of endogenous peroxidase varies significantly between tissues. Leukocytes exhibiting high endogenous peroxidase levels usually require longer incubation (45 min) than other tissues. If you encounter signals in your negative controls try to diminish those signals either by prolongation of the quenching reaction or by increasing the concentration of H 2 O 2 up to 3%. 14. Evaporation control is of the utmost importance for reliable and reproducible results as unspecific signals because of the evaporation of the reaction mixture may result from insuf-
442 Wiedorn and Goldmann ficient sealing. From the many methods for sealing, the use of Pattex Supermatic200Plus offers a convenient and reliable way to prevent leakage (15,29,30). It is easier to handle than nail polish with the same good sealing capacity. Furthermore, it seems to represent a biocompatible adhesive as it does not interfere with enzyme activity during PCR. 15. Coverslips can easily be removed by an initial scalpel cut along the sealed edge, which is to be lifted first. To facilitate the removal of the coverslips, the slides can be stored at 4°C for 1 to 2 min to completely harden the gluten if it is still viscous after the incubation at 92°C (8,15,29). 16. The lower the temperature and the shorter the incubation period, the better will be morphology which, nevertheless, is negatively affected by the IS-PCR procedure. 17. Temperatures, concentration of SSC, and incubation period have to be adjusted for the probe in use. 18. Antibody concentrations have to be adapted for different probes and according to the efficiency of the PCR. One can choose from a wide variety of chromogens. Furthermore, a peroxidase-based system can be used using AEC+ or DAB+ (Dako) as chromogens, which usually will result in more localized signals (8,28) than those achieved with NBT/BCIP. Detection solutions and antibody concentrations have to be adjusted. Acknowledgments The authors are indebted to Prof. Dr. Dr. E. Vollmer for making it possible to participate in the development of this technology and H. Kühl for excellent technical assistance. References 1. Chen, R. H. and Fuggle, S. V. (1993) In situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression. Am. J. Pathol. 143, 1527–1534. 2. Goldmann, T., Becher, B., Wiedorn, K. H., Pirow, R., Deutschbein, M. E., Vollmer, E., et al. (1999) Epipodite and fat cells as sites of hemoglobin synthesis in the branchiopod crustacean Daphnia magna. Histochem. Cell <strong>Bio</strong>l. 112, 335–339. 3. Heniford, B. W., Shum-Siu, A., Leonberger, M., and Hendler, F. J. (1993) Variation in cellular, E. G.F receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction. Nucleic Acids Res. 21, 3159–3166. 4. Long, A. A., Komminoth, P., and Wolfe, H. J. (1992) Detection of HI.V provirus by in situ polymerase chain reaction. N. Engl. J. Med. 327, 1529. 5. Nuovo, G. J. (1994) PCR in Situ Hybridization Protocols and Applications, Second Ed., Raven Press, New York. 6. Patel, V. G., Shum-Siu, A., Heniford, B. W., Wieman, T. J., and Hendler, F. J. (1994) Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction. Am. J. Pathol. 144, 7–14. 7. Snijders, P. J. F., van den Brule, A. J. C., Schrijnemakers, H. F. J., Snow, G., Meijer, C. J. L. M., and Walboomers, J. M. M. (1990) The use of general primers in the polymerase chain reaction permits detection of a broad spectrum of human papillomavirus genotypes. J. Gen. Virol. 71, 173–181. 8. Wiedorn, K. H., Kühl, H., Galle, J., Caselitz, J., and Vollmer, E. (1999) Comparison of insitu hybridization, direct and indirect in-situ PCR as well as tyramide signal amplification for the detection of HPV. Histochem. Cell <strong>Bio</strong>l. 111, 89–95. 9. Wiedorn, K. H., Goldmann, T., Kühl, H., Galle, J., and Vollmer, E. (1999) GenPoint : A new biotinyl tyramide based signal amplification system for significantly increasing sensitivity of in situ hybridization (ISH). Elec. J. Pathol. Histol. 994–999.
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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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Page 457 and 458: 470 Wang 2. Materials 2.1. PCR 1. D
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Page 473 and 474: 486 Preston amplification approach
Page 475 and 476: 488 Preston 1. 10× PCR buffer: 100
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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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