Redtaq ready mix pcr mix

All enzymes were purchased from Fermentas. Genomic DNA of T. forsythia WT strain ATCC 43037 was isolated from 2 ml of bacterial suspension as described previously and used as the DNA template in all PCRs, unless otherwise specified.^{37 (link)} The GeneJET™ Gel Extraction Kit (Fermentas) was used to purify DNA fragments from agarose gels and to purify digested plasmids and oligonucleotides. Plasmid DNA from transformed cells was isolated with the GeneJET™ Plasmid Miniprep kit (Fermentas). Agarose gel electrophoresis was performed as described elsewhere.³⁸ Primers for PCR and DNA sequencing were purchased from Invitrogen (Table 4). PCR was performed using the Phusion^® High-Fidelity DNA Polymerase (Fermentas) and a My CyclerTM (Bio-Rad) thermal cycler. Transformation of chemically competent E. coli DH5α and BL21 (DE) cells was performed according to the manufacturer’s protocol (Invitrogen). E. coli transformants were screened by PCR using RedTaq Ready-Mix PCR mix (Sigma-Aldrich) and recombinant clones were analyzed by restriction mapping. Expression vector and knockout cassette were sequenced (Microsynth) prior to transformation.

All enzymes were purchased from Thermo Scientific. Genomic DNA of P. alvei CCM 2051^T was isolated by using a Genomic Tip 100 kit (Qiagen) according to the manufacturer’s instructions, except that cells were broken by repeated freezing and thawing cycles (10 times) (Zarschler et al. 2009 (link)). The GeneJET™ Gel Extraction Kit (Fermentas) was used to purify DNA fragments from agarose gels and to purify digested plasmids and oligonucleotides. Plasmid DNA from transformed cells was isolated with the GeneJET™ Plasmid Miniprep kit (Fermentas). Agarose gel electrophoresis was performed as described elsewhere (Sambrook et al. 1989 ). Primers for PCR and DNA sequencing were purchased from Invitrogen (Table III). PCR was performed using the Phusion^® High-Fidelity DNA Polymerase (Fermentas) and the thermal cycler My CyclerTM (Bio-Rad). Transformation of chemically competent E. coli DH5α cells and E. coli BL21 (DE3) cells was done according to the manufacturer’s protocol (Invitrogen). Transformation of P. alvei CCM 2051^T wild type, P. alvei CCM 2051^T ΔwsfP, P. alvei CCM 2051^T ΔPAV2c_01630 and P. alvei CCM 2051^T ΔPAV2c_01640 cells is described elsewhere (Zarschler et al. 2009 (link)). Transformants were screened by PCR using RedTaq ReadyMix PCR mix (Sigma-Aldrich), and recombinant clones were analyzed by restriction mapping and confirmed by sequencing (Microsynth).