Phusion plus pcr master mix

Four different DNA Polymerase reagents were tested as part of this study: GoTaq Green Master Mix (Promega, Madison, WI, USA), Phusion Plus PCR Master Mix (Thermo Scientific, Waltham, MA, USA), AmpliTaq Gold Master Mix (Applied Biosystems, Waltham, MA, USA), and Hemo KlenTaq (New England BioLabs, Ipswitch, MA, USA). The gene chosen for amplification was the 16s rRNA gene and the primers used have been published previously [26 (link)]. Following PCR, the amplified DNA was run on a 1% agarose gel, stained with ethidium bromide, and imaged using a Bio-Rad Gel Doc EZ Imager (Bio-Rad, Hercules, CA, USA). Gel images are shown in Supplementary File S2. All reagents were used following protocols described by their respective manufacturers and run in a Bio-Rad T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). DNA extracted through the phenol–chloroform method yielded higher concentrations than the other two extraction methods; therefore, 0.5 µL of DNA from each sample was used for PCR. The exact protocol details and thermocycler conditions for each reagent are as follows:

Gene sequence variants coding PsLam81A (native enzyme sequence with C-terminal 6×His tag), PsLam81AΔCBM56 (truncated enzyme sequence without CBM56 domain with C-terminal 6×His tag) were cloned from genomic DNA of Paenibacillus sp. GKG using Phusion Plus PCR Master Mix (Thermo Fisher Scientific) and designed primers PsLam81A and PsLam81AΔCBM56 (Table S1). The PCR products were stained with ROTI Load DNA stain 1 SYBR Green (Carl-Roth, Karlsruhe, Germany) and ran in a 1% agarose gel electrophoresis. Bands corresponding to target gene sizes were excised from gel and plasmid DNA was extracted from gel slices using GeneJET Gel Extraction Kit (Thermo Fisher Scientific). Purified PCR products were cloned into a pLATE31 cloning vector using aLICator LIC Cloning and Expression Kit 3 (C-terminal His-tag) (Thermo Fisher Scientific, Vilnius, Lithuania). E. coli strain DH5α (Fermentas, Vilnius, Lithuania) cells were transformed with reaction mixtures and spread on LB agar plates supplemented with 75 µg/mL ampicillin. pDNA was extracted and purified using the ZR Plasmid Miniprep kit (Zymo Research, Irvine, CA, USA). pDNA was sequenced using LIC sequencing primers (Table S1) by the Sanger method (Macrogen Europe, Amsterdam, The Netherlands). Two additional sequencing primers PsLam81A-mid were designed for native enzyme sequence coding gene to obtain whole gene sequence coverage.

Excision assays were designed as described previously (45 (link)). Briefly, a multiplex PCR assay was designed to produce amplicons of distinct sizes if the Pf prophage was integrated (primers Fwd_1 and Rev produce a smaller band) or excised (primers Fwd_2 and Rev produce a larger band) using Phusion Plus PCR Mastermix (Thermo Scientific # F631L). Primers were used at a final concentration of 0.5 µM and are listed in (Table 3).

Genomic DNA was isolated from tail biopsies using the Purlink Genomic DNA mini kit (#K1820-02, Invitrogen, Waltham, MA, USA). The forward genotyping primer (F1-WT and KO) was the same for the WT and KO alleles: 5′-ACCAGATAAGGGTGGGGTTC-3′. The WT reverse primer (R1-WT) was 5′-GTGCCCCACAAAGGTCTCT-3′, generating a 675 bp product. The KO reverse primer (R2-KO) was 5′-TTGCTCCCGTGAGAGACTTT-3′, producing a 596 bp product. The PCR amplifications were performed with Phusion Plus PCR master mix (#F631S, ThermoFisher Scientific, Waltham, MA, USA) with amplification conditions being initial denaturation at 98°C (30 s), then 30 cycles of denaturation 98°C (10 s), annealing 57°C (10 s), extension 72°C (15 s), and a final extension of 72°C (5 min).