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RNA Arbitrarily Primed PCR 249 Fig. 1. RAP-PCR fingerprints. Products from RAP-PCR were resolved by electrophoresis as described. DNA molecular weights were determined using 32 P-labeled DNA fragments from MspI-digested pBR322 plasmid DNA, lane 1. Lanes 2 through 25 contain RAP-PCR products prepared from human fibroblast total RNA. Fingerprints were prepared from fibroblasts synchronized by serum starvation for 48 h and then treated by addition of serum containing media for the indicated times prior to isolation of total RNA, including isolation of total RNA from nonserum-starved cells. Fingerprinting of each sample was performed using three different concentrations of total RNA in the cDNA reaction, that is, 500, 250, and 125 ng per 10 µL of reaction. The fourth (empty) lane in each sample is the reverse transcriptase minus control.
250 Ringquist et al. 3. Purification of fluorescently labeled material from unincorporated dye was performed by centrifuging the sample in a microfuge using Microcon columns twice, using 500 µL TE (pH 8.0) washes each time, according to the manufacturer’s instructions. 4. Labeling of PCR products can also be performed by Terminal Transferase-catalyzed addition of Cy3-dCTP or Cy5-dCTP, as well as other fluorescent-labeled or modified deoxynucleotide triphosphates. 1 µg of PCR product in 20 µL was combined with 25 µL 2× terminal transferase buffer and 5 µL of 1 mM Cy5-dCTP or 1 mM Cy3-dCTP. The reaction was performed at 37°C for 1 h and purified using the Microcon columns as described previously. 5. After purification over microcon columns, fluorescent probe was dried under vacuum and dissolved in 8 µL of 1× TE buffer. Preparation of probe in hybridization solution was performed by addition of 8 µL of probe solution to 2 µL of blocking solution, 2.1 µL of 20× SSC, and 0.4 µL of 10% SDS. The solution was then incubated 1 min in a boiling water bath, and allowed to cool on the bench top for 30 min. 3.6. Microarray Hybridization 1. Preparation of microarrays followed the protocol of Eisen and Brown (16) and used polylysine-treated slides and PCR products generated from sequenced human cDNA libraries; please see Eisen and Brown (16) for details on preparing polylysine-coated slides. Inserts from the IMAGE library, supplied by Research Genetics, were PCR amplified by standard means using the vector-specific primers. Printing of PCR products was done using an Omnigrid robot to spot PCR products onto GoldSeal glass slides using a 16 pin print head. After printing, slides were rehydrated briefly over an 80°C waterbath and snap heated for about 6 s by placing the slides on an 80°C heat block. Ultraviolet treatment was performed using a Stratalinker 2400 (Stratagene, La Jolla, CA) at 60 mJ for 1 min. The slides were then baked at 80°C for 2 h and stored. 2. Immediately before hybridization, slides were treated with succinic anhydride as described by Eisen and Brown (16). The succinic anhydride blocking solution must be prepared fresh by placing 6 g of succinic anhydride into a dry beaker followed by addition of 335 mL of 1-methyl-2-pyrrolidinone. After the succinic anhydride was dissolved 15 mL of 1 M sodium borate, pH 8.0 was added. The solution was stirred for 5 s to mix the solutions. The mixture was then poured into a clean glass chamber and the slides placed quickly inside and incubated for 15 min. 3. Immediately after succinic anhydride blocking, slides were placed in a chamber with boiling water (the volume should be at least twice that of the blocking solution). The slides were plunged up and down and shaken for 2 min, and then the slides were immediately transferred to a chamber containing 95% ethanol, agitated and then dried for 37°C for 15 min. 4. Hybridization was performed by placing one glass chamber with distilled water in a 65°C oven for at least 30 min before doing the hybridization. One hybridization chamber was cleaned with pressurized air, and 2 drops (10 µL) of 3XSSC (450 mM sodium chloride, 45 mM sodium citrate, pH 7.0) were placed in the wells. The slides were then cleaned with pressurized air and placed in the hybridization chamber. The probe was then pipetted onto the slide, usually in 40 µL, taking care to avoid bubbles. The coverslip was then cleaned with pressurized air and placed over the probe. This volume of probe usually spreads easily along the surface; if not, the overslip can be pressed to help the spreading. The chamber was then sealed and submerged in a 65°C water bath for 4 to 16 h. 5. The hybridization was stopped by washing in 2× SSC, 0.1% SDS, followed by washing in 1× SSC. Samples were incubated for 2 min with 0.2× SSC. The washing steps were performed at room temperature. Drying should be avoided in all the washing steps. Prepare
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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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Page 195 and 196: 198 McDonagh the dilution method is
Page 197 and 198: 200 McDonagh 2.2. Basic PCR 1. dNTP
Page 199 and 200: 202 McDonagh Fig. 2. Quantitative P
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Page 203 and 204: 206 Bartlett Table 1 Example Assay
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Page 221 and 222: 226 Dominguez et al. Table 1 Primer
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Page 227 and 228: 232 Dominguez et al. 3.4. Gel Elect
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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
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Page 247 and 248: 252 Ringquist et al. 2. The present
Page 249 and 250: 254 Ringquist et al.
Page 251 and 252: 256 Case-Green, Pritchard, and Sout
Page 267 and 268: 272 Oien Fig. 1. A schematic diagra
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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
Page 283 and 284: 290 Edwards and Bartlett ing less p
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Page 289 and 290: 296 Rithidech and Dunn 11. Ethidium
Page 291 and 292: 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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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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