Protein Engineering Protocols - Mycobacteriology research center

178 Stebel et al.because of the additional work without significant benefit, the loss of material,and the higher error rate.3.6. Quantification of Purified FragmentsTo analyze and compare the yield of the purified DNA fragments, we initiallyquantified them with SYBR Green II. Because the NExT shuffling proceduregives highly reproducible amounts, we later omitted the quantification step (seeNote 16). We typically obtained a fragment concentration of 40 to 60 ng/µL.1. Stain 2 µL samples of the purified DNA fragments (see Subheading 3.4.) in 50 µLof 1:5000 diluted SYBR Green II, which is a highly sensitive dye for singlestrandedDNA (see Note 17).2. Incubate this mixture for 5 min in a dark box and measure the fluorescence (weused a Perkin Elmer Fluorescence Spectrometer LS 50B in 96-well format; seeNote 18). The dye is excited at 480 nm and the emission is measured at 515 nm.3. Use an oligonucleotide in the size range of your fragments in different dilutions asa calibration curve to determine fragment concentration.3.7. Gene Reassembly and AmplificationThe full-length gene is reassembled from the purified gene fragments (seeSubheading 3.5.) in an internal primer extension procedure with increasingannealing temperatures using, as the preferred choice, a proofreading DNA polymerase,such as Vent (Fig. 3). In the internal primer extension PCR, the fragmentsserve each other as primers and, thus, get longer with each cycle of thereaction, until full-length products are achieved. As a final step, products of theassembly reaction are amplified with a standard PCR with end primers, cloned,and sequenced. While establishing the method, the assembly reaction was monitoredby agarose gel electrophoresis (Fig. 3). The assembly process was stoppedafter an increasing number of cycles, and the products obtained at theses pointswere subjected to the amplification PCR. The underlying principle of the finalsteps is the same as in other gene assembly protocols (3), but there are importantchanges. Despite the harsh chemical cleavage conditions, the assembly worksvery efficiently and a proofreading polymerase was used.1. Use approx 2 µg of the purified DNA fragments (see Subheading 3.5.) for thereassembly. In our case, even less than 1 µg of DNA fragments was sufficientwhen using Vent polymerase. We normally used 20 to 25 µL of purified fragments,without measuring the concentration.2. Mix the DNA fragments with 4 µL (see Note 19) of a mixture of 10 mM of eachdATP, dTTP, dCTP, and dGTP (800 µM final), and 4 U Vent DNA Polymerase(NEB), with 1 to 4 µL of 25 mM MgSO 4and 5 µL of the supplied 10X buffer.Adjust the volume to 50 µL with water.3. Cycles for the reassembly are: one cycle of 94°C, 3 min; 36 cycles of 92°C, 30 sdenaturation; 30°C, 60 s + 1°C per cycle (cooling ramp 1°C/s) annealing; 72°C, 1 min+ 4 s per cycle extension; and final incubation at 72°C, 3 min.