Dna miniprep kit

DNA for transient transfection was prepared by Miraprep of E. coli DH5α cells as previously described using a Qiagen (Valencia, CA) DNA miniprep kit (Pronobis et al., 2016 (link)). Briefly, transformed E. coli DH5α cells were grown in 50 mL LB media supplemented with ampicillin (50 µg/mL) overnight at 37°C. Cells were collected by centrifugation and resuspended in P1 buffer supplemented with fresh RNase. After alkaline lysis and neutralization, the supernatant was cleared by centrifugation. The supernatant was diluted with an equal volume of 96% (v/v) ethanol prior to loading onto five Qiagen miniprep spin columns. At this point, the DNA was washed and eluted according to the Qiagen protocol. Purity of the eluted DNA was checked by measuring the A_{280 nm}/A_{260 nm} ratio on a NanoDrop 2000c spectrophotometer and by agarose gel electrophoresis. The correct gene sequence for p300 was also confirmed by sequencing prior to transfection in human cells.

3XFlagNICD1 was a gift from Raphael Kopan (Addgene plasmid # 20183; http://n2t.net/addgene:20183; RRID:Addgene_20183) [70 (link)]. Puro-iNotch1IC was a gift from Danwei Huangfu (Addgene plasmid # 75338; http://n2t.net/addgene:75338; RRID:Addgene_75338) [71 (link)]. EX-Z9294-M03 (human LRP8 expression construct) was purchased from GeneCopoeia (USA). Following transformation, plasmid DNA was extracted from host bacteria using a DNA Miniprep Kit (Qiagen, Australia) according to the manufacturer’s instructions and quantified by spectrophotometry using a NanoDrop (Thermo, Australia).

The selected LAB strains were molecular characterized by 16S ribosomal RNA gene sequencing analysis. Total DNA was extracted from the LAB strains using a Qiagen DNA mini prep kit (Qiagen Korea Ltd., Jung-Gu, Seoul, Korea) according to the manufacturer’s guidelines. A purified DNA sample was sent to Bioneer Corporation, Korea, for 16S-rRNA gene amplification and sequencing. The obtained 16S-rRNA gene sequences were compared to those of known Lactobacillus strains in the NCBI-BLAST database, and the authenticity of the strains was confirmed.

Total RNA was extracted from 5 controls and 8 PD midbrain samples using Trizol (Invitrogen, MA, USA), and genomic DNA was extracted from 4 controls and 3 PD samples using the DNA miniprep kit (Qiagen, Venlo, Netherlands). The mRNA coding regions, spanning 423 bp, were fully covered. The genomic location was designed to amplify a 219-nucleotide-long DNA template containing the S42Y and A53E regions. AmpliconEZ from GENEWIZ was used for NGS sequencing using the following primers: cDNA Forward-ATGGATGTATTCATGAAAGGACTTTC, cDNA Reverse-GGCTTCAGGTTCGTAGTCTT, gDNA Forward-ACTAGCTAATCAGCAATTTAAGGCT, and gDNA Reverse-TGTTCTTAGAATGCTCAGTGATTG. PCR amplification was performed using high-fidelity DNA polymerase (Platinum Superfii PCR master mix; Invitrogen, MA, USA). The presence and size of each amplicon were confirmed by gel electrophoresis using a 2% agarose gel. Amplified fragments were subsequently purified using the PCR Purification Kit (Thermo Scientific, MA, USA). The double-stranded DNA (dsDNA) was quantified using Qubit, normalized to a concentration of 20 ng/µL, with a total amount of 500 ng. Following DNA library preparation, sequencing reactions and data processing by GENEWIZ, over 300,000 reads were aligned to the reference sequence for low-frequency variant detection. The variant allele frequency (VAF) cutoff was set at 0.1%.

The strains Achromobacter sp. 4(2010), Pseudomonas stutzeri strain 9 and Rahnella sp. strain EK12 were isolated from hydrocarbon-contaminated soil samples from the Polish Carpathian Mountains. The isolated strains were phenotypically characterized using the standard techniques (gram staining, colony shape, size and colour on nutrient agar plate, catalase and oxidase test, etc.), according to Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994 ). Bacterial DNA was isolated from the pure culture using the DNA Mini Prep Kit (Qiagen, Venlo, The Netherlands). For 16S rRNA gene amplification, the bacteria-specific primers 8F 5′AGTTTGATCATCGCTCAG 3′ and 1492R 5′GGTTACCTTGTTACGACTT3′ were used (Lonergan et al. 1996 (link)). The 16S rRNA gene sequence determined in this study was deposited in the GeneBank database of NCBI under the accession numbers HM246520.1 (Achromobacter sp. 4(2010)), JN006140.1 (P. stutzeri strain 9) and JQ409469 (Rahnella sp. strain EK12).

293T cells were transfected with the appropriate siRNAs. At 24 h after siRNA transfection, pSP189 plasmids were irradiated with UV (1000 J/m²) and transfected into the cells using Lipofectamine 2000 (Invitrogen). Cells were harvested 48 h later for plasmid purification using the DNA miniprep kit (QIAGEN). The purified plasmids were digested with DpnI and transformed into the bacterial strain MBM7070 by electroporation. Bacterial cells with wild-type SupF tRNA expressed functional β-galactosidase and formed blue colonies on X-gal plates, whereas bacteria with mutated SupF formed white colonies. The mutation frequency in the SupF gene was analyzed by counting the ratio between blue (wild-type) and white (mutant) colonies. Mutations in the SupF gene were confirmed by DNA sequencing.