Mastercycler pcr system

E. faecium was grown in BHI medium, bacterial DNA was extracted using alkaline lysis, and the EntF-encoding DNA fragment was amplified in a Mastercycler PCR system (Eppendorf, Belgium). Each reaction was prepared in 10 μL (final reaction volume) containing 2x BioMix (Bioline, Belgium) and 1 μL of DNA 0.5 μM of each primer (EntF*-PCR primers, Additional file 5: Fig. S2b). Amplification was performed as follows: 1 cycle of 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. Final elongation was performed at 72 °C for 10 min, after which PCR products were kept at 4 °C. The PCR amplification products were resolved on 1.5% agarose gel.

The 16S rRNA genes were amplified from previous bacterial DNA using conventional PCR with primers 16S-F (5′-CCAGCAGCCGCGGTAATACG-3) and 16S-R (5′-ATCGG (C/T) TACCTTGTTACGACTTC-3′) [7 (link)], producing an amplicon of about 996 bp. PCR amplifications were performed using 5 L of template, 5 L of 10 × PCR buffer, 4 M of each primer stock solution, 4 mM of each dNTP, 1.25 U of ex Taq DNA polymerase (TaKaRa), and sterile distilled water added to a final total volume of 50 L. Amplification was performed using a Mastercycler® PCR System (Eppendorf International, Hamburg, Germany). Thermocycling parameters were 94°C for 10 min, 35 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 2 min; a final extension step at 72°C was added for 5 min. The products of PCR amplification were examined by gel electrophoresis [100 V through a 1.5% agarose gel with 0.5 × TBE (Tris-borate-EDTA) running buffer], stained with ethidium bromide, and analyzed with the GelDoc XR Gel Documentation System (Bio-Rad, USA.). PCR amplicon sizes were estimated by comparison to molecular size markers (TaKaRa) then purified and sequenced (Sanger capillary sequencing) at the Beijing Genomics Institute (Shenzhen, China).

The peri-infarct tissues that were supplied by the MCA were excised from the brain tissue on ice, snap-frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated using TRIzol reagent (Takara, Dalian, China) according to the instructions of the manufacturer. With a PrimeScript RT Reagent kit (Takara), 1 μg of RNA was reverse transcribed, and genomic DNA was eliminated by the addition of DNase. The primers for the PCR assays were supplied by Sangon Biotech (Shanghai, China) and were as follows: claudin-5, 5′-GGCGATTACGACAAGAAGAACT-3′ (sense) and 5′-CCCGAACCCAACCTAACTT-3′ (antisense); β-actin, 5′-CCCATCTATGAGGGTTACGC-3′ (sense) and 5′-TTTAATGTCACGCACGATTTC-3′ (antisense). RNA was quantified using the QuantiFast SYBR-Green PCR kit (Qiagen). The PCR assays were performed in an Eppendorf Mastercycler PCR system according to the following protocol: 5 min hold at 95°C, followed by 10 sec at 95°C and 30 sec at 60°C (40 cycles). The transcript levels were standardized with β-actin and calculated using the ΔΔCT method.

Total RNA was extracted from the lyophilized and ground mycelium using a TRIzol kit (Invitrogen, CA, USA), and it was then treated with RNase-free DNase (Takara Inc, Dalian, China) to remove possible contaminant DNA. The first-strand cDNA was generated by reverse transcription in a 20-μl reaction using a Moloney Murine Leukemia Virus (M-MLV) RTase cDNA synthesis kit (Takara Inc.). qRT-PCR was performed on a Mastercycler PCR system (Eppendorf, Germany) using SYBR green as a fluorescence reporter (BioRad, CA, USA) following the manufacturer’s protocol. Reactions were set up based on three replicates per sample. Controls without the addition of the templates were included for each primer set. The PCR cycling parameters were as follows: pre-incubation at 94 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 15 s, annealing at 59 °C for 30 s, and extension at 72 °C for 32 s. The expression of each gene of interest (expressed as the Ct value) was normalized against β-actin mRNA. The method has been described previously [30 (link)]. The qRT-PCR data were analyzed using the 2^−ΔΔCt relative quantification method [36 (link)]. The primers for RT and PCR are provided in Additional file 1: Table S1.

ABCC1 specific primers spanning different regions (Fig. 1a) were generated using Primer-BLAST on the ABCC1 cDNA sequence from Ensembl genome browser ENST00000399410.8 ABCC1-202 (matching the NCBI Reference sequence NM_004996.4 formerly NM_004996.3). Different combinations of primers were used for PCR using Extender PCR to Gel Mastermix (Amresco]). The amplicons were then run on a 2% agarose gel stained with ethidium bromide. PCR was done using the Mastercycler PCR System (Eppendorf).

Restriction enzymes (New England Biolabs), Taq DNA polymerase (MP Biomedicals), and Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) were used as recommended by the manufacturers. Oligonucleotides were purchased from Eurogentec and are listed in Table S2. PCRs were performed with a Mastercycler PCR system (Eppendorf). DNA sequences were determined by GATC Biotech. Electrotransformation of L. lactis was performed as described previously (37 (link)).