Repli g mini kit

For whole-genome sequencing, a total of nine DNA extracts from seven equine placental and foetal samples from the epizootic, a parrot tissue sample from central NSW and C. psittaci CR394 isolate from a Crimson Rosella parrot from Blue Mountains endemic region were treated with the NEBNext Microbiome DNA Enrichment kit (New England Biolabs, Ipswich, Massachussets, USA) to deplete host methylated-DNA, followed by the Agencourt AMPure XP Bead Clean up kit (Beckman Coulter, Brea, California, USA) according to the manufacturer’s instructions. Samples were then subjected to multiple displacement amplification (MDA), using the Qiagen Repli-G mini kit (Qiagen, Australia) to increase the yield of bacterial DNA. All samples were quantified for C. psittaci genome copy number prior to and following MDA using a C. psittaci-specific qPCR assay targeting a 263 bp fragment of the C. psittaci-specific ORF_O607 gene using C. psittaci F3 and B3 primers^{24 (link)}.

We used Qiagen REPLI-g Mini Kit (QIAGEN) containing φ29 DNA polymerase. 5 ul fresh Buffer D1 was added to the 5 ul circular DNA sample above, incubated at room temperature for 3 min. 10 ul fresh Buffer N1 was then added to the mix to stop the reaction. Then put 30 ul prepared REPLI-g DNA Polymerase master mix (29 ul REPLI-g Mini Reaction Buffer and 1 μL REPLI-g Mini DNA Polymerase) into the sample, and incubated the reaction at 30 °C for 16 hours. After heat inactivate the reaction at 65 °C for 3 minutes, the quantity of circular DNA obtained was measured by a Qubit Fluorometer.

Locus-specific primers were prepared for amplification of the Agro_gc40, Agro_gc50 and Agro_gc60, BtKIT1-10 and BtKIT27-37 sequences (Additional file 1), and PCR was performed with Phusion Hot Start DNA polymerase (Thermo Fisher Scientific) and The Expand Long Range dNTPack (Roche Applied Science, Penzberg, Germany), following the manufacturer’s protocol. 5-methyl-dCTP (TriLink, CA, USA) was added to the PCR mixture at final concentrations of from 0 to 60 μM. WGA was performed using the REPLI-g mini kit (QIAGEN). Following the manufacturer’s protocol, 2.5 μl DNA (12.5-75 ng) was denatured with the D1 solution for three minutes, and then neutralised with the N1 solution. The amplification was performed in the reaction mixture with the presence of from 0 to 100 μM 5-methyl-dCTP (final concentrations) at 30°C for 16 hours. After incubation, the DNA polymerase was heat-inactivated, and the products were diluted with the same amount of water.

Ryegrass endophyte genomic DNA was amplified with the QIAGEN REPLI-g mini kit. A section (2-3 mm²) of endophyte mycelium was placed into 6 μl PBS solution. The Buffer D2 (7 μl) was added into the sample and incubated on ice for 10 minutes, following mixing by a vortex. The Stop Solution was, then, added and mixed by a vortex. The reaction mixture, consisting of 29 μl REPLI-g Reaction Buffer, 1 μl REPLI-g Mini DNA Polymerase and 1 μl 5-methyl-dCTP (750 μM), was added into 9 μl of denatured sample, and the sample was incubated at 30°C for 16 hours. The amplicons (5 μl) were digested with MspJI and the sequencing library preparation was performed following the BAC clone sequencing protocol. The PCR-enriched DNA was cleaned with AMPure XP bead solution (x0.8). The sequencing library was characterised with the Agilent 2100 Bioanalyzer and Qubit® Fluorometer.

Arabidopsis BAC clones (MIXK3, F20D21 and F20B17) were amplified with the QIAGEN REPLI-g mini kit. Glycerol stock (4 μl) of a BAC-containing E. coli clone was mixed with the Buffer D1 (4 μl) and incubated on ice for 5 minutes. The Buffer N1 (8 μl) was added into the sample and mix by stirring with a tip. The sample was incubated at room temperature for 3 minutes. The reaction mixture, consists of 5.8 μl REPLI-g Reaction Buffer, 0.2 μl REPLI-g Mini DNA Polymerase and 0.4 μl 5-methyl-dCTP (750 μM), was added into 3.4 μl denatured sample, and the reaction mixture was incubated at 30°C for 16 hours. The amplicons (5 μl) were digested with MspJI and the end-filling reaction was performed with Klenow Fragment (3’ → 5’ exo^-). Sequencing adopter ligation was performed with T4 ligase (NEB) and ligated DNA was cleaned with AMPure XP bead solution (x0.8) to exclude short DNA. DNA fragments were subsequently enriched through PCR with the phusion DNA polymerase Kit. Small fragments (<500 bp), in which fraction E. coli genome-derived fragments were highly prevalent, were removed through size-selection with AMPure XP bead solution (x0.6). The sequencing library was characterised with the Agilent 2100 Bioanalyzer, Agilent DNA 1000 Kit, and the Qubit® Fluorometer (Life Technologies), following the manufacturer′s protocols.

Uninjected control zygotes and injected surplus zygotes are cultivated in NCSU-23 HEPES base medium, supplemented with cysteine and BSA at 38.5°C for 5–7 days. Blastocysts were harvested at day 7 post cultivation and the genome amplified using the REPLI-g Mini Kit (Qiagen), according to the manufacturer’s instructions. Genotyping was performed as described below.