Protein Engineering Protocols - Mycobacteriology research center

176 Stebel et al.for preparative gels (see Subheading 3.5.2.). Gels have to be prepared freshlybecause old gels do not run properly due to urea degradation. We used 31.5 g urea,29.2 mL of 30.8% (37, 5:1) acrylamide:bis-acrylamide, 17.1 mL H 2O, 7.5 mL of10X TBE, 500 µL of 10% ammoniumperoxodisulfate, and 50 µL TEMED; sufficientfor nine analytical gels in one gel casting unit (see Note 8).2. Gels are run during the whole procedure at 56°C. We used a Hoefer Mighty smallbasic unit (Amersham) with an attached temperature-controlled water bath.3. Prerun the gels in 1X TBE buffer for 10 min at 100 V.4. Concentrate the cleaved DNA before loading in a speed-vac to approx 7 µL to evaporatethe piperidine, supplement it with 25 µL of deionized formamide and heat it to80°C for 3 min in a thermocycler (see Note 9). The samples thus prepared can besupplemented with 3 µL of 60% sucrose solution and 7 µL H 2O to aid sample loading(see Note 10). Fifteen to 20 µL of this mixture (~3 µg DNA) were loaded.5. Mixtures of oligonucleotides as well as a 100-bp ladder can serve as length standard(see Note 11). Mix at least one marker with dye-containing loading buffer.6. Run the loaded gel at 170 V until the bromphenol blue added to the marker isapproximately 0.5 cm from the end of the gel (see Note 12).7. Stain the gel for 5 min in 30 mL of a 1 µg/mL ethidium bromide solution (1:5000dilution) and visualize the DNA in UV light (see Note 13).Analytical urea gels revealed that the fragmentations yielded size distributionswith defined peaks, depending on the dUTP fraction. As seen in Fig. 1B,several length distributions were easily obtained, including pools of very smalland large fragments, which is optimal for shuffling short genes or long geneclusters, respectively. Such tests were required initially to determine the optimaldUTP fraction for obtaining the desired length distribution for a given gene.Once established, the desired dUTP fraction of 33.3% for the test libraries wasused and found to be highly reproducible. Thus, the gel analysis step was nolonger required and omitted for subsequent rounds of directed evolution.3.5. Fragment PurificationGene fragments resulting from the chemical or enzymatic cleavages (seeSubheading 3.3.) were cleaned either directly from the cleavage reaction solution,using a silica-based resin (Subheading 3.5.1.), or over a preparative denaturingpolyacrylamide urea gel (Subheading 3.5.2.).Initially, we thought that gel purification was crucial to permit only fragmentsof finite size, and to ensure that no long fragments or even full-length genes wereinvolved in the reassembly reaction; a potential problem that could have loweredthe crossover frequency. However, no full-length product could be amplifiedfrom the directly purified fragments without several cycles of the reassemblyprocess, demonstrating a very efficient fragmentation (see Subheading 3.7.).Thus, purification from gels is only necessary when a very narrow size rangeis required.