Q5 high fidelity dna polymerase

For the assembly of 509 sequences, the oligo pool was synthesized and the lyophilized pool consisted of 11,776 oligos of 192 nts (synthesized by Twist Bioscience), which included the 152 nts payload in each oligo. The pool was resuspended in 1× TE buffer for a final concentration of 2 ng/μL. One of the files, 509 oligos, was flanked by binding sites for the premixed primers F02/R02. PCR was performed using Q5® High-Fidelity DNA Polymerases (NEB #M0491) and primers F01-F04/R01-F04 (10 ng oligos, 2.5 μL of each primer mix (100 mM), 0.5 μL Q5 High-Fidelity DNA Polymerase, 4 μL 2.5 mM dNTPs in a 50 μL reaction). Thermocycling conditions were as follows: 5 min at 98 °C; 10 cycles of: 10 s at 98 °C, 30 s at 56 °C, 30 s at 72 °C, followed by a 5 min extension at 72 °C. The library was then purified using Plus DNA Clean/Extraction Kit (GMbiolab Co, Ltd. #DP034P) and eluted in 40 μL ddH₂O. This library was considered the master pool and run on a 2% agarose gel to verify the correct size. For the assembly of 11520 sequences, the synthetic DNA pool consisted of 11520 oligos of 200 nts (synthesized by Twist Bioscience), which included the 155 nts payload flanked by binding sites for the primers F1/R1 (Supplementary Fig. 3). The lyophilized pool was rehydrated in 1× TE buffer and the above protocol was used to amplify the file.

The broad-host shuttle-vector system pMTL80000 (Heap et al., 2009 (link)) was used for all cloning steps. All generated plasmids of this study (Supplementary Table 1) were cloned with restriction endonucleases and T4 ligase (New England Biolabs, Frankfurt am Main, Germany) or Gibson assembly (NEBuilder^® HiFi DNA Assembly, New England Biolabs, Frankfurt am Main, Germany). PCR work was carried out with primers provided by IDT (Integrated DNA Technologies, Iowa, United States) (Supplementary Table 2) and with a proof-reading Q5^® High-Fidelity DNA Polymerase (New England Biolabs, Frankfurt am Main, Germany)) according to the manufacturer’s guidelines. Genomic DNA (gDNA) was purified from 2 mL of exponential cultures of C. ljungdahlii with the NucleoSpin Tissue Mini kit (Macherey-Nagel, Düren, Germany) and used as PCR-template. Notably, instead of performing harsh cell disruption according to the manufacturer’s recommendation, we applied a 6 × 10 s vortex interval during the procedure. All PCR steps were performed with Q5^® High-Fidelity DNA Polymerase (New England Biolabs, Frankfurt am Main, Germany) and primers provided by IDT (Integrated DNA Technologies, Iowa, United States) (Supplementary Table 2). PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Hilden, Germany).

To construct luciferase-based reporter plasmids containing observed mutations in LTR regulatory elements, an overlap extension PCR was used to amplify each particular LTR variant. The first round of PCR allowed for the amplification of two sequences encompassing the LTR: a 5’-end fragment of 979 bp and 3’-end fragment of 571 bp were generated using Q5 High-Fidelity DNA Polymerase and Q5 Reaction Buffer (New England BioLabs, MA, USA) at the following thermal conditions: 30 s at 98 °C, 40 cycles (10 s at 98 °C, 45 s at 65 °C (5’-end fragment) or 52 °C (3’-end fragment), 1 min at 72 °C), and 7 min at 72 °C. The resulting amplicons were subjected to a second PCR using P8169 and P520 oligonucleotides described in Table S3 and Q5 High-Fidelity DNA Polymerase (New England BioLabs, MA, USA), as described above. Thermal conditions were as follows: 30 s at 98 °C, 38 cycles (10 s at 98 °C, 50 s at 64 °C, 1 min 15 s at 72 °C), and 9 min at 72 °C. To construct Tax expression plasmids, the tax gene was amplified by nested PCR using the oligonucleotide primers described in Table S3. First round PCR was performed as described in Section 2.2. Nested PCR was carried out using Q5 High-Fidelity DNA Polymerase with the following thermal conditions: 30 s at 98 °C, 35 cycles (10 s at 98 °C, 25 s at 58 °C, 1 min at 72 °C), and 10 min at 72 °C.