Working cultures of Ng isolates used in Fig 4 were prepared as described in “Ng culture preparation,” and 1.5 mL of the cultures were pelleted at 2,500g for 2.5 min and resuspended in 150 μL normal human urine (Lee Biosciences) prewarmed to 37°C. Initial exposure was performed by incubating 100 μL control and treated samples at 37°C in PCR tube strips on a Bio-Rad C1000 Thermal Cycler. Treated samples consisted of 65 μL MHB, 5 μL NaHCO3 (100 mM), 5 μL DNase I (2 U/μL), 5 μL PEN (20 μg/mL), and 20 μL Ng isolate suspension in urine. NF-H2O was used in place of PEN in control samples. A CHAPS enhancing step was performed as described previously. After the enhancement step, a 20-μL aliquot from each sample was extracted by diluting 5X in QuickExtract DNA Extraction Solution (Lucigen) and heated for 1 min at 65°C followed by 1 min at 98°C on a Bio-Rad C1000 Thermal Cycler. All sample handling following antibiotic exposure was performed using a multichannel pipette. Amplification was then performed using qPCR, ddPCR, or dLAMP. Extractions were diluted 2.5X in NF-H2O before use in dLAMP.
C1000 thermal cycler
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The C1000 Thermal Cycler is a laboratory equipment designed for conducting polymerase chain reaction (PCR) experiments. It is capable of precisely controlling and cycling the temperature of samples to facilitate the amplification of DNA or RNA sequences.
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975 protocols using c1000 thermal cycler
1
Antibiotic Exposure and DNA Extraction of Ng Isolates
2
Quantitative PCR for Neisseria gonorrhoeae
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Working cultures of Ng isolates were prepared as described in “Ng culture preparation.” Incubation at 37°C was performed in 100 μL reaction volumes in PCR tube strips on a Bio-Rad C1000 Thermal Cycler. Treated samples consisted of 77.5 μL MHB, 2.5 μL NaHCO3 (200 mM), 5 μL DNase I (2 U/μL), 5 μL PEN (20 μg/mL), and 10 μL working Ng isolate culture. PEN was replaced with NF-H2O in control samples. A 10-μL aliquot of each sample was extracted at 15, 30, 45, 60, 90, and 120 min and diluted 10X in QuickExtract DNA Extraction Solution (Lucigen, Middleton, WI), then heated for 6 min at 65°C followed by 4 min at 98°C on a Bio-Rad C1000 Thermal Cycler. All sample handling following antibiotic exposure was performed using a multichannel pipette; qPCR and calculation of % accessibility were performed as described previously.
3
Thermal Stability Analysis of Protein Constructs
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20 μL 25 μM BLA construct in PBS with 100 mM DTT was incubated at 10, 25, 37, 55, 75, 90 or 100 °C for 10 minutes and then cooled to 10 °C on a Bio-Rad C1000 Thermal Cycler. 20 μL 10 μM phytase construct in 50 mM Tris-HCl pH 7.0 with 2 mM CaCl2 was incubated at 10, 25, 55, 75, 90 or 100 °C for 10 minutes and then cooled to 10 °C on a Bio-Rad C1000 Thermal Cycler. The ramp rate was 3 °C/s. Samples were spun at 17,000 g at 4 °C for 30 minutes and the supernatant was removed for SDS-PAGE. Every gel had a triplicate loading control to normalize the samples. Loading samples consisted of the protein sample that had been kept at 10 °C. The loading control was defined as 100% soluble fraction. Bands were quantified using densitometry values obtained from the Image Lab 3.0 software.
4
Real-Time PCR Detection of Toxoplasma gondii
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The individually synthesized cDNA was analyzed in pools of four with 0.5 μl of cDNA from each tick. The primers and probe (Invitrogen Corporation) target the B1 gene of T. gondii [22 ], its function is unknown, but it is present in all investigated genotypes of T. gondii. The positive control used was T. gondii DNA [445 ng/µl]. It was kindly provided by professor Krzysztof Solarz through Dr Olga Pawelczyk, both the Department of Parasitology, Medical University of Silesia, Poland. Detection limit was determined at [445 pg/µl].
One reaction contained 18 µl of master mix and 2 µl sample cDNA. The master mix contained 6.3 of µl RNase/DNase free water, 10.5 µl of Maxima Universal Master Mix (Thermo Fisher Scientific), 0.4 µl of T. gondii forward primer [5 µM], 0.4 µl of T. gondii reverse primer [5 µM], 0.4 µl of TaqMan probe [10 µM]. A period of 30 s at 50°C was added to every cycle before the 15 s of denaturation. A C1000 thermal cycler (Bio-Rad) was used for the real-time PCR.
The PCR assay consisted of one initializing denaturation at 95°C for 10 min and then 40 cycles of 95°C denaturation for 15 s and 60°C Annealing/Elongation for 60 s. A C1000 thermal cycler (Bio-Rad) was used for the real-time PCR.
One reaction contained 18 µl of master mix and 2 µl sample cDNA. The master mix contained 6.3 of µl RNase/DNase free water, 10.5 µl of Maxima Universal Master Mix (Thermo Fisher Scientific), 0.4 µl of T. gondii forward primer [5 µM], 0.4 µl of T. gondii reverse primer [5 µM], 0.4 µl of TaqMan probe [10 µM]. A period of 30 s at 50°C was added to every cycle before the 15 s of denaturation. A C1000 thermal cycler (Bio-Rad) was used for the real-time PCR.
The PCR assay consisted of one initializing denaturation at 95°C for 10 min and then 40 cycles of 95°C denaturation for 15 s and 60°C Annealing/Elongation for 60 s. A C1000 thermal cycler (Bio-Rad) was used for the real-time PCR.
5
Quantitative Real-Time PCR Analysis
6
Total RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted from the freshly enucleated eyeball using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc. Waltham, MA, USA), according to the manufacturer's instructions. cDNA was generated using the PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) at 37°C for 15 min, followed by 85°C for 5 sec and then maintained at 4°C. RT-qPCR was performed according to the manufacturer's instructions using a Bio-Rad C-1000 Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primer sequences are listed in Table I . All primers were purchased from Invitrogen (Thermo Fisher Scientific, Inc.).
RT-qPCR was performed in a 20 µl reaction mixture using the FastStart Universal SYBR-Green Master reagent (Roche Diagnostics) on an ABI PRISM 7000 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR conditions were as follows: 1 cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. The 2−ΔΔCq method was used to estimate the relative transcript levels (22 (link)). The levels of GAPDH amplification were used to normalize each sample Cq value. Units are expressed as relative quantification (n=6 per group).
RT-qPCR was performed in a 20 µl reaction mixture using the FastStart Universal SYBR-Green Master reagent (Roche Diagnostics) on an ABI PRISM 7000 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR conditions were as follows: 1 cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. The 2−ΔΔCq method was used to estimate the relative transcript levels (22 (link)). The levels of GAPDH amplification were used to normalize each sample Cq value. Units are expressed as relative quantification (n=6 per group).
7
Quantitative Analysis of Drug Resistance Genes
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Total RNA was extracted from harvested cells with TRIzol (Thermo Fisher Scientific). Briefly, for the detection of Bcl-2, MRP3, MRP7, and P-glycoprotein, 1 µg of total RNA per sample was converted to cDNA using a cDNA Synthesis Kit (Thermo Fisher Scientific). The cDNAs were amplified and detected using a SYBR Green PCR kit (Qiagen). GAPDH was used as an endogenous control. For detection of the mRNA, cDNA products were synthesized using the miScript Reverse Transcription Kit (Qiagen). Sequence-specific primers targeting Bcl-2, MRP3, MRP7, P-glycoprotein, or the endogenous control U6 were purchased from Qiagen (Table 1 ). Quantitative reverse-transcription PCR (qRT-PCR) was performed using miScript SYBR Green PCR kit (Qiagen). All reactions were run in triplicate on a Bio-Rad C1000 thermal cycler (Bio-Rad). mRNA expression fold changes were calculated according to the 2−ΔΔCT method.
8
Quantification of miRNA and mRNA Levels
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Total RNA from cells, tissues and exosomes were isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. RNA content was measured using a Nanodrop-2000 (Thermo Fisher Scientific, Waltham, MA, USA). One microgram of RNA was employed to synthesize cDNA using the PrimeScript RT Reagent Kit Perfect Real Time (TaKaRa, Dalian, China) or the miScript II RT Kit (Qiagen, Hilden, Germany). To determine the amount of the individual miRNA and mRNA levels, we employed the fluorescent quantitative real time PCR (qRT-PCR) using the Fast SYBR Green Chemistry (TaKaRa, Dalian, China) on Bio-Rad C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA). All the primers listed in Supplementary Table S3 were obtained from AuGCT Co. (Beijing, China). The reactions were performed in a 20 μl volume in triplicate with the following amplification steps: an initial denaturation step at 95°C for 3 min, followed by 44 cycles of denaturation at 95°C for 10 s, anneal at 56°C for 15 s and extension at 72°C for 15 s. Finally, the relative quantities of miRNA and mRNA were calculated using 2−ΔΔCT method after normalization to RNU6B or GAPDH or miR-16.
9
Quantitative Analysis of p53, p21 and MDM2 Expression
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Gene expression of p53, p21 and MDM2 was determined by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). After treatment at IC50 concentrations as described above, total RNA was extracted with the Ambion Minikit (Ambion, Carlsbad, CA) and cDNA was synthesized (High Capacity cDNA Archive kit, Applied Biosystems, Carlsbad, CA) according to manufacturer’s instructions. Using TaqMan primer sets for p53, p21 (cyclin-dependent kinase inhibitor 1), and MDM2, qRT-PCR was performed in triplicate with the housekeeping gene cyclophilin (PPIA; Applied Biosystems) as normalizer. The Bio-Rad C1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) was used for all reactions, and fold-change was calculated with the 2-△△Ct method.
10
Molecular Sequencing of Malaria Genes
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Nested PCR products were purified with ExoSAP (New England Biolabs, Ipswich, MA, USA). The cycling sequencing was carried out using Big Dye Terminator v3.1 cycle sequencing kit on Bio-Rad C1000 thermal cycler (Bio-Rad, Hercules, CA, USA). Dye terminators were cleaned by precipitating reactions with EDTA/NaOAc/Ethanol precipitation and rehydrating in 10 μl HiDi formamide. Capillary electrophoresis was performed in ABI 3730 or 3730xl genetic analyzer (Applied Biosystems, Foster City, USA). Sequence data were analysed using the Geneious Prime R10 and R11 (Biomatters, San Francisco, CA, USA). The mixed SNPs were identified where the minor peak was ≥ 30% of the major peak. SNPs at Pfdhfr, Pfdhps, Pfcrt, Pfmdr1 genes and Pfk13 propeller region were identified by comparing sequences with reference 3D7 strains from GenBank XM_001351443.1, Z30654.1, XM_001348968.1, XM_001351751 and MW712622.1), respectively. PCR reaction systems and cycling conditions were detailed in Additional files 1 , 2 .
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