Pcr master mix

The genomic DNA of patients was extracted from whole blood using standard methods (QIAamp, QIAGEN, Hilden, Germany). The −94ins/delATTG NF-κB1 insertion–deletion (−94ins/delATTG) promoter polymorphism (rs28362491) was determined restriction analysis. Therefore, the primer NF-κB1 insertion–deletion (−94ins/delATTG) polymorphism _del/ins_SE (5′-CTTGGATCCATGCCGACCC-3′) and NF-κB1 insertion–deletion (−94ins/delATTG) polymorphism _del/ins_AS (5′-TAGGGAAGCCCCCAG-GAAG-3′) were used to amplify a 158 bp PCR fragment, followed by digestion with restriction enzyme PFiMI (New England Biolabs, Ipswich, PA, USA). The PCR was performed at an annealing temperature of 60 °C in a 25 µL commercially available PCR master mix (New England Biolabs). The analysis was performed using agarose gel electrophoresis (PeqLab, Erlangen, Germany), resulting in a 158 bp fragment for D-allele and a 121 bp product for I-allele. Randomly chosen samples with known genotype were reanalyzed for result verification.

The genes in Space-3 payload included several cold stowage items: R9.4 flow cells and reagents, including control DNA, Rapid 16S sequencing kits SQK-RAS201 (Oxford Nanopore Technologies (ONT), Oxford, UK), PCR master mix and exonuclease I (New England BioLabs (NEB), Ipswich, MA, USA), all aliquoted into spaceflight-certified tubes. A pipette kit including 20, 200, and 1000 μL Eppendorf Research Plus pipettes (Eppendorf Hamburg, Germany) with associated individually packaged tips was stowed at ambient temperature. The hardware was launched from Cape Canaveral Air Force Base on the Orbital ATK OA-7 Cygnus resupply vehicle on 18 April 2017. All reagents were launched and maintained at −90 °C, while flow cells were stowed and launched at + 4 °C. Upon docking with the ISS, the flow cells and reagents were transferred to the refrigerator (+2 to + 8 °C) and freezer (−80 to −90 °C) dewars within the Minus Eighty Degree Laboratory Freezer for ISS (MELFI), respectively.

Based on the user manual, total genomic DNA was extracted from donkey stool samples using the Magnetic Soil and Stool DNA Kit (Tiangen Biochemical Technology, Beijing). DNA was extracted from fecal samples using the cetyltrimethylammonium bromide (CTAB) method, and DNA stress and concentration were detected using 1% agarose gel electrophoresis. Amplification was performed using Phusion High-Fidelity polymerase chain reaction (PCR) Master Mix (New England Biolabs; 341F CCTAYGGGRBGCASCAG, 806R GGACTACNNGGGTATCTAAT). The mixed and purified PCR products were then constructed through terminal repair and street sequencing. NovaSeq 6,000 was used to perform onboard sequencing on PE 250. Clean reads were obtained by excluding low-quality and barcode reads.

Bacterial DNA was extracted by boiling 1 mL of overnight culture, as described by Parvin et al. [7 (link)]. Extended-spectrum beta-lactamase gene grouping (blaTEM, blaSHV, blaCTX-M-1, and blaCTX-M-2) was performed by multiplex polymerase chain reaction (PCR) using primer set and PCR conditions [7 (link)]. In brief, amplification reactions were set in a 25-μL volume containing 12.5 μL of PCR master mix (New England Biolabs, Massachusetts, USA), 1.0 μL (10 pmol) of each of the forward and reverse primers, 1 μL of DNA, and 3.5 μL of nuclease-free water. Polymerase chain reaction was run using T100 thermal cycler (Bio-Rad Laboratories, Inc., California, USA) with multiplex PCR conditions: initial denaturation at 95°C for 5 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. Appropriate positive and negative controls (sterile phosphate buffer saline) were included in each PCR run. The PCR products were visualized by electrophoresis on a 1.5% agarose (TaKaRa, Shiga, Japan) gel containing ethidium bromide. The DNA bands were photographed using a UV transilluminator (Cell Biosciences, Victoria, Australia).

To recover the lineage barcodes, we used an aliquot of the excess full-length cDNA generated in the 10x library protocol. Specifically, we selectively amplified reads containing the lineage barcode using primers that flank the 10x cell barcode and the end of the lineage barcode in our library (Supplementary Table 1)⁶⁹ . To perform the PCR, we combined 100ng of full-length cDNA per reaction, 0.5µM of each primer, and PCR master mix (NEB, M0543S). We used 12 cycles to amplify the cDNA using the following protocol: an initial 30 s denature step at 98 °C, then 98 °C for 10 s followed by 65 °C for 2 min repeated 12 times, and a 5-min final extension step at 65 °C. We then extract the amplified barcodes, which are ~1.3 kb, using SPRI beads (Beckman Coulter, B23317) for size selection (0.6X bead concentration). To sequence the barcode library, we used a NextSeq 500 with a Mid Output Kit v2.5 (150 cycles, Illumina, 20024904). We performed paired-end sequencing and used 28 cycles on read 1 to read the 10x barcode and UMI, 8 cycles on each index, and 123 cycles on read 2 to sequence the lineage barcode.

A uniplex PCR targeting methicillin resistance gene (mecA) in S. aureus was standardized, and used in this study with specific primer as described earlier [27 (link)]. The sequence of the forward primer was MecA1 (5′-GTAGAAATGACTGAACGTCCGATAA-3′) and of the reverse primer was MecA2 (5′-CCAATTCCACATTGTTTCGGTCTAA-3′) for the amplification of 310 bp. Each PCR reaction mixture was constituted in a final reaction mixture of 25 µL made up of 12.5 µL PCR master mix (New England BioLabs Inc., Ipswich, MA, USA), 1.5 µL (15 pmol) each of forward and reverse primers, 7.5 µL of nuclease-free water, and 2 µL of DNA template. Amplification was performed by using a Veriti 96-well thermal cycler (Thermo Fisher Scientific) with this thermal profile: heating at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min, extension at 72 °C for 2 min, and a final extension step for 10 min at 72 °C. Positive (mecA gene) and negative (sterile phosphate buffer saline) controls were included in each run of PCR. After amplification, PCR product was subjected to electrophoresis on 1.5% UltraPure™ agarose gel containing ethidium bromide (0.5 μg/mL). The resulting band of PCR product was examined under UV-transilluminator and photographed. A 100-bp molecular weight standard ladder was included on each run.

Gibson Assembly was carried out using the Gibson Assembly master mix described by DG Gibson et al. (Gibson et al., 2009 (link)). Plasmid and DNA gene blocks were mixed in a 1:3 ratio in a Gibson Assembly master mix and incubated at 50°C. E. coli BL21 cells were transformed with the assembled plasmids and plated on LB agar medium with kanamycin at 50 μg/mL. Selected colonies were added to the NEB PCR master mix with 2.5 μL of corresponding primers. PCR was carried out as per NEB Q5 polymerase PCR protocol. Sanger sequencing (GATC light-run) was also performed on the selected colonies to confirm the assembly (Eurofins Genomics, UK Ltd.).