Clonejet cloning kit

Primers were designed to amplify hldD, hldD/waaF, and hldD/waaF/waaC, including a region of 310 base pairs upstream of hldD. The forward primer was the same for each construct (5’-ACG AGT CGT GTC GAG TTG TTG TTC-3’). The following reverse primers, specific for each construct were used: 5’- CAT GGC CTA ACG GCA TTG GAA TTG-3’ for hldD, 5’- CGT CAG CGC GGG TAA GGT ATG TAA A-3’ for hldD/waaF, and 5’- AGC ACC ACA TTG AGC CGT GGT TAT-3’ for hldD/waaF/waaC. PCR products were cloned using the CloneJET cloning kit (Fermentas) and transformed into DH5α cells. Plasmids were isolated from ampicillin resistant clones using the QIAprep Spin Miniprep Kit (QIAGEN) and then cycle sequenced to confirm amplification of the proper gene and also to ensure that the inserts were in the appropriate hldD/waaF/waaC orientation relative to the lac promoter sequence. Once this was confirmed, complementation plasmids were transformed into the hldD::Tn5 mutant strain background via electroporation.

The total DNA from each soil sample was obtained using a SPIN Kit for Soil® (MP Biomedicals), according to the manufacturer's instructions. 16S rRNA gene was partially amplified (variable regions 1 and 2) with Y1 and Y2 primers [16 (link)], yielding Polymerase Chain Reaction (PCR) products of approximately 300 bp. The PCR reaction mixture consisted of 20–50 ng/μL of DNA, 5.0 pmol/μL of each primer, 0.2 mM of dNTPs, 3.0 mM of MgCl₂, Buffer 1x, and 2.5 U Taq DNA polymerase (Ludwig Biotec). A thermocycler model PTC-100™ Programmable Thermal Controller (MJ Research, Inc.) used a thermal profile of 95°C for 2 min, 35 cycles of 95°C for 45 s, 65°C for 45 s, and 72°C for 1 min, and 30 s ending with 72°C for 5 min. After the PCR reaction, the products were purified with a Wizard® SV Gel and PCR Clean-Up System (Promega) and for PCR cloning CloneJET™ Cloning Kit (Fermentas) was used as per the manufacturer's instructions.
Colonies in Petri dishes were collected with a sterile wooden toothpick and organized into 96-well ELISA plates containing 100 μL of Luria-Bertani medium with ampicillin (50 μg/mL). After overnight development of the clones at 37°C, a copy of each library was inoculated to extract the plasmid DNA [17 ] and 100 μL of 40% glycerol (v/v) was added to the original library for storage at −80°C.