Protein Engineering Protocols - Mycobacteriology research center

NExT DNA Shuffling 1773.5.1. Direct Purification From Piperidine Cleavage1. Fragments are most easily purified directly from the piperidine cleavage (seeSubheading 3.3.) using the QiaexII kit (Qiagen) according to the manufacturer’smanual (see Note 14). Add the capture buffer included and neutralize (~20 µL of3 M acetate buffer).2. After two washing steps, extract the fragments from the matrix by adding 25 µLelution buffer in two steps. Pool the two elutions.3. Centrifuge two times, transferring to a fresh tube each time (see Note 15).3.5.2. Purification From Denaturing Polyacrylamide Urea GelsNote that this step is optional. For the fragment extraction from urea gels,two methods were tested. Either the classic water extraction with subsequentacetate/Mg 2+ precipitation or the procedure recommended by Qiagen for theirQiaexII kit were used. Both methods suffered from loss of material not extractedfrom the gel, as assessed by test stainings of already extracted gel material.1. For the extraction of fragments from preparative polyacrylamide urea gels (seeSubheading 3.4., step 1), cut out the desired range (Fig. 2B) with a scalpel whilevisualizing bands on a low-intensity UV table. Crush the gel piece thoroughly in atube and incubate it with either diffusion buffer (QiaexII extraction, step 2) or 1 mLwater (water extraction, step 3) in a thermomixer (Eppendorf) at 37°C, 1000 rpmovernight.2. Fragments extracted with diffusion buffer are purified with the QiaexII kit, asdescribed in Subheading 3.5.1.3. Precipitate the water-extracted oligonucleotides by adding 1/10 volume of acetatebuffer, 1/100 volume of MgCl 2, and 1 volume of 2-propanol; incubate at –20°C for1 h; and centrifuge for 15 min at 20,000g at 4°C. Resuspend the pellet in 50 µL 90%ethanol in a thermomixer at 37°C, 1000 rpm for 1 h; incubate for 1 h at –20°C; andcentrifuge for 15 min at 20,000g at 4°C. Air-dry the pellet and dissolve it in 30 µLelution buffer by incubating in a thermomixer at 37°C, 1000 rpm for 1 h.To elucidate the crossover rate with a gel extraction step, a test shufflingexperiment with three parental clones (CAT_Nd10 mutants) was set up. Two ofthe clones contained one and one clone contained two distinct mutations withina stretch of 100 bp. Sequencing of eight clones shuffled and assembled with Taqpolymerase revealed that six clones had one crossover within the 100-bpstretch. Thus, the crossover rate is in the range of the shuffling procedure usingthe quick clean up (see Subheadings 3.5.1. and 3.8.). A total of 3851 baseswere sequenced, and 12 errors were found, equaling a mutation rate of 0.31%.The error rate is significantly higher than with the quick clean up, which mightbe explained by UV damage caused by visualization even on the weak 366-nmlight source used and/or chemical modifications caused by the gel. For the preferredNExT DNA shuffling method, we omitted the gel purification step,