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Cloning PCR Products 391 8. Wang, K., Koop, B. F., and Hood, L. (1994) A simple method using T4 DNA polymerase to clone polymerase chain reaction products. Biotechniques 17, 236. 9. New England Biolabs catalog (1994) Reference Appendix, pp. 180,181. 10. Kaufman, D. L. and Evans, G. A. (1990) Restriction endonuclease cleavage at the termini of PCR products. Biotechniques 9, 304. 11. Jung, V., Pestka, S. B., and Pestka, S. (1993) Cloning of polymerase chain reactiongenerated DNA containing terminal restriction endonuclease recognition sites, in Methods in Enzymology, vol. 218 (Wu, R., ed.), Academic, London, pp. 357–362. 12. Pfeiffer, B. H. and Zimmerman, S. B. (1983) Polymer-stimulated ligation: Enhanced bluntor cohesive-end ligation of DNA or deoxyribonucleotides by T4 DNA ligase in polymer solutions. Nucleic Acids Res. 11, 7853–7871.
392 Walsh
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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
Page 331 and 332: 340 Stirling concentrations interfe
Page 333 and 334: 342 Daniels to sequence a 500-bp PC
Page 335 and 336: 344 Daniels 4. Load all of the samp
Page 337 and 338: 346 Daniels References 1. Orita, M.
Page 339 and 340: 348 Kösel et al. Fig. 1. Schematic
Page 341 and 342: 350 Kösel et al. 3. Methods 3.1. P
Page 343 and 344: 352 Kösel et al. Fig. 2. Sequencin
Page 345 and 346: 354 Kösel et al. 5. Kwok, S. and H
Page 347 and 348: 356 Mazars and Theillet Fig. 1. Sch
Page 349 and 350: 358 Mazars and Theillet of genomic
Page 351 and 352: 360 Mazars and Theillet
Page 353 and 354: 362 Suomalainen and Syvänen Fig. 1
Page 355 and 356: 364 Suomalainen and Syvänen detect
Page 357 and 358: 366 Suomalainen and Syvänen temper
Page 359 and 360: 368 Shen et al. ladder. Also, direc
Page 361 and 362: 370 Shen et al. 3. Mineral oil. 4.
Page 363 and 364: 372 Shen et al. extended primers, w
Page 365 and 366: 374 Quivy and Becker Fig. 1. The us
Page 367 and 368: 376 Quivy and Becker that decreases
Page 369 and 370: 378 Quivy and Becker 2. Solution of
Page 371 and 372: 380 Quivy and Becker 7. Precipitate
Page 373 and 374: 382 Quivy and Becker 7. Prepare a p
Page 375 and 376: 384 Quivy and Becker
Page 377 and 378: 386 Walsh by most, but not all M13
Page 379 and 380: 388 Walsh PCR products are now flus
Page 381: 390 Walsh 5. For palindromic restri
Page 385 and 386: 394 McAleer, Coffey, and Dunham Fig
Page 387 and 388: 396 McAleer, Coffey, and Dunham Tab
Page 389 and 390: 398 McAleer, Coffey, and Dunham Tab
Page 391 and 392: 400 McAleer, Coffey, and Dunham
Page 393 and 394: 402 Stirling 5. Homopolymer Regions
Page 395 and 396: 406 Speel, Ramaekers, and Hopman Ta
Page 397 and 398: 408 Speel, Ramaekers, and Hopman Fi
Page 399 and 400: 410 Speel, Ramaekers, and Hopman po
Page 401 and 402: Table 2 Comparison of PRINS, in Sit
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Page 405 and 406: Table 3 Summary of Control Experime
Page 407 and 408: 418 Speel, Ramaekers, and Hopman 3.
Page 409 and 410: 420 Speel, Ramaekers, and Hopman ad
Page 411 and 412: 422 Speel, Ramaekers, and Hopman 25
Page 413 and 414: 424 Speel, Ramaekers, and Hopman 63
Page 415 and 416: 426 Bull and Paskins Fig. 1. Result
Page 417 and 418: 428 Bull and Paskins 2.3. Detection
Page 419 and 420: 430 Bull and Paskins 6. Shake the s
Page 421 and 422: 432 Bull and Paskins
Page 423 and 424: 434 Wiedorn and Goldmann Fig. 1. IS
Page 425 and 426: 436 Wiedorn and Goldmann 41. Protei
Page 427 and 428: 438 Wiedorn and Goldmann 2. Incubat
Page 429 and 430: 440 Wiedorn and Goldmann 3.15. Prim
Page 431 and 432: 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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458 Speel, Ramaekers, and Hopman 3.
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460 Speel, Ramaekers, and Hopman 4.
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462 Speel, Ramaekers, and Hopman bu
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464 Speel, Ramaekers, and Hopman 26
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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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