Genejet dna gel extraction kit

To insert the Eco105I endonuclease restriction site into the appropriate location at the hairpin loop following the domain-connecting α-helix of TconTS, sense and reverse primers were designed annealing at this target location and comprising the Eco105I site (S1 Table). DNA sequences coding for TconTS-CD-α-helix with the Eco105I site attached at the 5’-end were amplified using HindIII sense primer in combination with the corresponding Eco105I reverse primer (S1 Table). In addition, DNA sequences encoding the corresponding TconTS-LD sequence with the Eco105I site attached to the 3’-end were amplified using BamHI reverse and the appropriate Eco105I sense primer (S1 Table). Both PCR products were digested using Eco105I Fast Digest restriction enzyme (Thermo Scientific, Germany), purified using GeneJET DNA Gel Extraction Kit (Thermo Scientific, Germany) and blunt-end ligated using T4-DNA ligase, following instructions of the manufacturers, to generate full TconTS sequence with the Eco105I restriction site inserted. Appropriate TconTS DNA sequences were cloned into modified pET28aMBP expression vector using BamHI and HindIII restriction enzymes according to manufacturers instructions. All sequences and insertions were confirmed by DNA sequencing at the Max Planck Institute for Marine Microbiology, Bremen, Germany.

Rat faeces samples were processed using the E.Z.N.A.^® Stool DNA Kit (Omega Bio-tek, Norcross, GA, USA) to extract total microbial genomic DNA. Before being used, the DNA quality and concentration were assessed using 1.0% agarose gel electrophoresis analysis and a NanoDrop^® ND-2000 spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA). An ABI GeneAmp^® 9700 PCR thermocycler (Thermo Scientific Inc., Waltham, MA, USA) was used to amplify the hypervariable region V3-V4 of the bacterial 16S rRNA gene using primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Each sample was amplified in triplicate.
The PCR products were run on a 2% agarose gel and then purified through the GeneJET DNA Gel Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA). The quantification was performed using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions.