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Recovery of PCR Products from Gels 77 18 Technical Notes for the Recovery and Purification of PCR Products from Acrylamide Gels David Stirling 1. Introduction Although the best way of obtaining pure polymerase chain reaction (PCR) product will always be to optimize reaction conditions to yield only one product, there are still circumstances where DNA has to purified from gels. Several good commercial products exist for the recovery of DNA from agarose. Here, we present a reliable method of recovering DNA from polyacrylamide gels. 2. Materials 1. Sterile scalpel blade. 2. Microfuge tubes (0.5 and 1.5µL). 3. Sterile narrow-gauge needle (23-guage). 4. Tris-HCl EDTA buffer: 10 mM Tris-HCl, 10 mM EDTA, pH 8.0. 5. 3 M sodium acetate (pH 4.0). 6. 100% ethanol. 7. 70% ethanol. 3. Method 1. Stain the gel with ethidium bromide, visualize the band of interest, and dissect with scalpel blade. 2. Pierce hole in the bottom of a 0.5 ml Microfuge tube with narrow gauge needle. 3. Place 0.5 mL of TE in a 1.5-mL microfuge tube. 4. Place the acrylamide slice with the dissected DNA band into a 0.5-mL tube (with hole) and place inside the 1.5-mL tube (the lip will prevent it touching the bottom). 5. Microfuge the two-tube combination at 12,000g for 5 min. The acrylamide will be forced through the hole in the bottom of the 0.5-mL tube into the TE. 6. Discard the empty 0.5-mL tube. 7. Cap the 1.5-mL tube, vortex for 30 s, then incubate for 2 h overnight at 37°C. 8. Microfuge at 12,000g for 5 min. 9. Transfer supernatant to fresh tube. 10. Add 1/10 volume 3 M sodium acetate and 2 volumes 100% ethanol. From: Methods in Molecular <strong>Bio</strong>logy, Vol. 226: PCR Protocols, Second Edition Edited by: J. M. S. <strong>Bartlett</strong> and D. Stirling © Humana Press Inc., Totowa, NJ 77
78 Stirling 11. Store at –20°C for at least 30 min. 12. Microfuge at 12,000g for 20 min at room temperature. 13. Discard supernatant and wash pellet in 70% ethanol. 14. Dry pellet and redissolve in 25 µL of TE. 15. If concentration of DNA is insufficient for further processing, re-amplify 1 µL in a second round of PCR.
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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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8 Carroll and Casimir of PCR. Such
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18 McDonagh stored. This means that
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22 Stirling which will either not a
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136 Benjamin, Smith, and Waites amp
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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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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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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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186 Cremer and Moos Fig. 1. Rearran
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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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212 Kerr Fig. 1. Fluorescent probes
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226 Dominguez et al. Table 1 Primer
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236 Dominguez et al. 11. Joshi, C.
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272 Oien Fig. 1. A schematic diagra
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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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288 Edwards and Bartlett technology
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292 Edwards and Bartlett Stepwise m
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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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310 Schmerer the investigation conc
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312 Schmerer References 1. Kunkel,
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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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324 Stirling 9. TAE (20×): 484 g o
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342 Daniels to sequence a 500-bp PC
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386 Walsh by most, but not all M13
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402 Stirling 5. Homopolymer Regions
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452 Gilchrist and Befus References
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470 Wang 2. Materials 2.1. PCR 1. D
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474 Wang
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486 Preston amplification approach
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492 Preston 2. Remove the AmpliTaq
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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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530 Burke and Barik purified mutant
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532 Burke and Barik
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