After treatment, cells were harvested and total DNA was extracted and separated by agarose gel electrophoresis after staining DNA with Ethidium bromide and documented under gel documentation system (UVP Multidoc-It).
Multidoc it
Manufactured by Analytik Jena
Sourced in United States
The MultiDoc-It is a versatile laboratory equipment designed for document imaging and capture. It provides high-quality scanning and archiving capabilities for a variety of laboratory documents and materials.
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Lab products found in correlation
Gelred, by Biotium (2 mentions)
Mirpremier microrna isolation kit, by Merck Group (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Rotor robot, by Singer Instruments (1 mentions)
Fast sybr green master mix kit, by Thermo Fisher Scientific (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Trizol reagent method, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 micro volume, by Thermo Fisher Scientific (1 mentions)
Bcip nbt, by Merck Group (1 mentions)
Nanodrop, by Eppendorf (1 mentions)
9 protocols using multidoc it
1
DNA Extraction and Agarose Gel
2
Quantitative High-Throughput Screening of Yeast Strains
3
Primer Validation for T. vaginalis
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The specificities of the primer pairs were verified by endpoint PCR, using cDNA from T. vaginalis trophozoites as the template and the enzyme Q5™High-Fidelity DNA polymerase (New England, BioLabsinc, Beverly, MA, USA), with the following amplification conditions: 30 s at 95 °C; 30 cycles of 10 s at 95 °C, 30 s at 61 °C, and 30 s at 72 °C; finally, 10 min at 72 °C. The amplified fragments were separated by electrophoresis in a 2% (w/v) agarose gel, dyed with GelRed (Nucleic Acid Gel, Biotium, Fremont, CA, USA), and visualized on a MultiDoc-It (UVP). To confirm the specificity of the primers and determine the efficiency of the PCR reaction, RT-qPCR analysis was carried out on a StepOne™ Real-Time PCR System and a Fast SYBR® Green Master Mix Kit (Applied Biosystems, Foster City, CA, USA), applying a 5-fold serial dilution consisting of five concentrations of cDNA, beginning at 100 ng; we then constructed standard curves to determine amplification efficiencies (E) for each candidate reference gene. Once the reaction cycles were completed, the melting curves for each gene were obtained, and the reactions were heated in a temperature range of 60 °C to 95 °C.
4
Small RNA Extraction and Purification
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Total RNA of the WB strain and transformant cells was extracted at different growth times by using the TRIZOL reagent method (InvitrogenTM). RNA was treated with DNAse I (Thermo Scientific) to prevent DNA contamination of the samples; RNA concentration and purity were then determined using a μDropTM plate (Thermo Scientific).
The extraction of small RNAs was performed with the mirPremier® microRNA Isolation Kit (Sigma-Aldrich), following the instructions of the manufacturer’s protocol. The small RNA obtained was electrophoresed in 3 % (w/v) agarose gels and visualized with GelRed (Nucleic Acid Gel, Biotium; Hayward, CA, USA) on a MultiDoc-It (UVP; Upland, CA, USA).
In addition, RNAs with sizes ≈ 100 bp were cut from acrylamide gels and purified using the aforementioned kit, following the protocols of Malone et al. [31 (link)] and Nielsen [32 (link)], with some modifications.
The extraction of small RNAs was performed with the mirPremier® microRNA Isolation Kit (Sigma-Aldrich), following the instructions of the manufacturer’s protocol. The small RNA obtained was electrophoresed in 3 % (w/v) agarose gels and visualized with GelRed (Nucleic Acid Gel, Biotium; Hayward, CA, USA) on a MultiDoc-It (UVP; Upland, CA, USA).
In addition, RNAs with sizes ≈ 100 bp were cut from acrylamide gels and purified using the aforementioned kit, following the protocols of Malone et al. [31 (link)] and Nielsen [32 (link)], with some modifications.
5
High-Resolution Plate Imaging Protocol
6
Cloning and Expression of H. pylori zwf Gene
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The Puc57 vector carrying the H. pylori zwf gene was digested using the NdeI and BamHI enzymes. The resulting digestion was analyzed in 1.0% agarose gel via electrophoresis and was subsequently stained with Midori Green Advance, then, finally, analyzed using the MultiDoc-It (UVP) equipment. The fragment (zwf gene) comprising 1278 base pairs (bp) was purified using a GeneJET Gel Extraction Kit, following the manufacturer’s protocol. Afterward, the gel fragment was purified and ligated into the expression vector pET3aHisTEVP, to generate pET3aHisTEVP-zwf (Figure S1 ). The resulting vector was sequenced in the Unidad de Síntesis y Secuenciación from the Instituto de Biotecnología, UNAM, Cuernavaca City Morelos. The results were compared with the BLAST sequencing database (NCBI), resulting in 99 hits with 100% identity and an E-value of 0.0 for H. pylori. The pET3aHisTEVP-zwf plasmid containing the zwf gene insert was used to transform E. coli BL21(DE3)Δzwf:kanr.
7
Total RNA Extraction and Small RNA Isolation
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Total RNA was extracted using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The concentration and the purity of the isolated RNA were quantified using a NanoDrop ND-1000 Micro-Volume (NanoDrop Technologies, Wilmington, DE, USA), and its integrity was verified in a 0.8% (w/v) agarose gel under denaturing conditions.
The extraction of small RNAs was performed with the mirPremier® microRNA Isolation Kit (Sigma-Aldrich, St. Louis, MO, USA), following the instructions of the manufacturer’s protocol. The small RNA obtained was electrophoresed in 3% (w/v) agarose gels and visualized with GelRed (Nucleic Acid Gel, Biotium; Hayward, CA, USA) on a MultiDoc-It (UVP; Upland, CA, USA).
In addition, RNAs with sizes ≈100 bp were copurified from total RNA and obtained by the aforementioned kit, following the protocol of Malone et al. [37 (link)] and Nielsen [38 (link)], with some modifications for this work.
The extraction of small RNAs was performed with the mirPremier® microRNA Isolation Kit (Sigma-Aldrich, St. Louis, MO, USA), following the instructions of the manufacturer’s protocol. The small RNA obtained was electrophoresed in 3% (w/v) agarose gels and visualized with GelRed (Nucleic Acid Gel, Biotium; Hayward, CA, USA) on a MultiDoc-It (UVP; Upland, CA, USA).
In addition, RNAs with sizes ≈100 bp were copurified from total RNA and obtained by the aforementioned kit, following the protocol of Malone et al. [37 (link)] and Nielsen [38 (link)], with some modifications for this work.
8
Analyzing Cellular Stress Responses
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After treatment, total protein was isolated from the cells and separated in 10% polyacrylamide gels, and transferred to nitrocellulose membrane (Pall). A positive control set for autophagy was induced by serum starvation for 3 h following the protocol from Zhang et. al. 2016. After transfer, membranes were incubated with primary antibodies [p53, AMPK, Caspase 3, Caspase 9, PARP1 (1:500); p62, S6, Beclin1 (1:750); LC3B, γ-actin (1:10,000)] for overnight at 4 °C. After washing, alkaline phosphatase (AP) conjugated secondary antibodies were added and incubated for 2 h. NBT-BCIP (Sigma) was used as substrate to develop the bands. Results were photographed by gel documentation system (UVP Multidoc-It) and densitometric analysis was done by NIH-ImageJ1.52A software.
9
Transcriptome Analysis by RT-PCR
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Tri regent was used to isolate total RNA from the cells as depicted from the manufacturer’s protocol. RNA was quantified by Nanodrop (Eppendorf) and bleach gels were used to check the integrity of the RNA samples43 (link). First strand cDNA synthesis kit was employed to prepare cDNA using 1 µg RNA as starting material as per manufacturer’s protocol. With the help of Primer 3 software,primersrequired for the PCR reactions were designed, using FASTA sequences of target genes from NCBI. The primer sequences and PCR profiles were listed in (Table S5 ).GAPDH was used as internal control. PCR products were separated in 2% Agarose gels and Gel documentation system (UVP MultiDoc-It) was used to photograph them. Densitometric analysis was done by Image J software.
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