Protein Engineering Protocols - Mycobacteriology research center

NExT DNA Shuffling 187staining. Because the uridine-incorporating PCRs for the radioactive and ethidiumbromide-stained gels were carried out with two different buffers (seeSubheading 2.2.), the addition of PCR enhancers, such as ammonium sulfate,Tween-20, or Triton X-100 have no significant effect on the dUTP vs dTTP incorporationrate.4. Notes1. Although the overall error rate of the described NExT DNA shuffling is alreadyquite low, the uridine-exchange PCR could, in addition, be performed with aproofreading polymerase. However, the polymerase should be selected carefullyto be able to sufficiently incorporate uridine and not to stall at uracil sites in thetemplate. Vent exo + (New England Biolabs) and nonuracil-stalling mutants of Pfupolymerase (Stratagene) have been reported to incorporate uridine.2. Very small fragments can be visualized radioactively. In this case, add 0.5 µL of a3.3 µM 32 P-dCTP solution (approx 0.5 µCi) to the uridine-exchange PCR mixture.For radioactive experiments, a PCR cleanup kit was used without the agarose gelstep (see Subheading 3.2.4.), because the nonradioactive template does not disturbvisualization of radioactive fragments.3. We used four 50-µL uridine-exchange PCR vials because our thermocycler worksbest with this volume.4. It is necessary to separate the uracil-containing products from nonuracil-containingtemplate, to make sure that no template is carried over.5. We preferred to use two columns in parallel to save time. In this case, we did theenzymatic and chemical cleavage of the uridine-exchange PCR (see Subheading3.3.) separately for the two tubes to keep the volume in a reasonable range for ourthermocycler. The samples can be combined for the fragment purification either atSubheading 3.5.1, step 2. or Subheading 3.4., step 4.6. Digests of up to 2 h or more yielded equivalent results, indicating a selective andconsistent digest.7. Piperidine is toxic and should be handled in a fume hood. All other solutions containingpiperidine, e.g., the used capture buffer of the Qiaex II kit (seeSubheading 3.5.1.), should be handled with care, and discharged in a closed tube.8. We used gels up to a maximum of 3 d after preparation. The urea powder adds tothe volume, which is taken into account in the final concentrations. The volumefor nine gels was used to pour eight gels.9. Piperidine influences the running properties of fragments and, thus, has to beevaporated as much as possible.10. For the radioactive experiments, only 7 µL of the DNA–formamide sample wereused, supplemented with 3 µL of 60% sucrose solution and 7 µL H 2O; 15 µL wereloaded onto the gel.11. For radioactive experiments, oligonucleotides and DNA ladder were kinased with32 P-γATP and purified by size exclusion to remove excessive 32 P-γATP. We usedSephadex G-50 material, but any nucleotide removal kit should serve this purpose.