Genomic DNA was extracted from etiolated leaves according to the CTAB method [61 ] with minor modifications and used as a template to amplify the set of full length 1Ay genes by priming the reactions via the degenerate pair PF1: 5’-ATGGCTAAGCGGC/TTA/GGTCCTCTTTG-3’, and PR1: 5’-CTATCACTGGCTAA/GGCCGACAATGCG-3’. Since the target sequences are known to have a high GC content, a high-fidelity ExTaq polymerase (Takara, Dalian, China) was used for PCR amplifications. The PCR amplifications were performed in a reaction volume of 50 μL containing 5 μL of 10 × ExPCR buffer, 0.2 mmol L−1 of dNTPs, 1 μmol L−1 of each primer, 2.5 U of ExTaq polymerase, and ddH2O to 50 μL. PCR was carried out using a Mastercycler pro PCR (Eppendorf, Hamburg, Germany). The cycling program consisted of 94 °C for 5 min, followed by 28 cycles of 94 °C for 40 s, 68 °C for 6 min, and a final extension at 72 °C for 10 min [19 (link)]. The amplified PCR products were visualized by 1.0% agarose gel electrophoresis followed by staining with ethidium bromide. The PCR amplification was performed in triplicate for each sample.
Pcr mastercycler pro
Manufactured by Eppendorf
Sourced in Germany
The Eppendorf PCR Mastercycler® pro is a versatile thermal cycler designed for performing polymerase chain reaction (PCR) experiments. It offers precise temperature control and reliable performance to ensure consistent and accurate results. The device features a compact and user-friendly design, making it a reliable solution for PCR-based applications in various laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Gel doc eq system, by Bio-Rad (2 mentions)
Ex taq polymerase, by Takara Bio (1 mentions)
Beyort 2 first strand cdna synthesis kit, by Beyotime (1 mentions)
Pcr master mix, by Beyotime (1 mentions)
Sub cell gt cell, by Bio-Rad (1 mentions)
Superscript 3 one step rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 real time pcr system, by Roche (1 mentions)
Sk br 3, by Korean Cell Line Bank (1 mentions)
Mcf 7, by Korean Cell Line Bank (1 mentions)
Mda mb 231, by Korean Cell Line Bank (1 mentions)
Mda mb 453, by Korean Cell Line Bank (1 mentions)
Hcc1937, by Korean Cell Line Bank (1 mentions)
Macsquant vyb, by Miltenyi Biotec (1 mentions)
Sybr green 1 dye, by Lonza (1 mentions)
Genomic dna clean concentrator kit, by Zymo Research (1 mentions)
7 protocols using pcr mastercycler pro
1
Amplification of 1Ay Genes from Etiolated Leaves
2
Validating HSP70 and Lysozyme in Regenerated Tissue
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To validate the up-regulated expression of HSP70 and lysozyme in 3-day regenerated tissue, 300 tail-amputated E. fetida were equally divided to 6 groups for different regeneration days (0-day, 12-h, 1-day, 2-day, 3-day and 4-day). RNA isolation was performed in each group with the same instructions in RNA-Seq analysis. cDNA was synthesised using BeyoRT™ II cDNA first strand synthesis kit (Beyotime Institute of Biotechnology, China) according to manufacturer's instructions. A final 20 μL cDNA was generated from 4 μg of RNA per sample. PCR amplification was performed with 0.1–1 μg final concentration of cDNA template, 0.8 μM Primer Mix (Table 6 ) and 1⨯ PCR Master Mix (Beyotime, China) on Mastercycler Pro PCR (Eppendorf AG., Germany) using the following cycling condition: 94 °C for 3 min, 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, followed by 72 °C for 10 min. The PCR products were analyzed on 1% agarose gel (Beyotime, China) with Sub-Cell GT cell (Bio-Rad Laboratories, Inc., U.S.A) under 100 V for 40 min. The results were analyzed by ImageJ version k 1.45 with the reference gene β-actin and the ratio of target genes/β-actin was calculated. A paired Student's t-test by using Prism 6 software was performed to determine the significance between different groups (P < 0.05) from three individual experiments.
3
Sensitive Detection of Dengue Viruses
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Detection of the four dengue virus serotypes in Ae. aegypti larvae and adults was modified from the method described by Tuksinvaracharn et al. [11 ]. The genomic viral RNA was extracted from pooled larvae and mosquitoes using the Invisorb® Spin Virus RNA Mini Kit (Invitex Gmbh, Germany) according to the manufacturer’s protocols. One-step RT-PCR was performed with five oligonucleotide primers (D1 and four type-specific primers, including TS1, TS2, TS3, and TS4) that were designed by Lanciotti et al [24 (link)]. Amplification was carried out in a 25 μl total mixture using the Superscript III one-step RT-PCR kit (Invitrogen, USA) with 10 μM of each primer and 6 μl of RNA. The RT-PCRs were performed in a PCR Mastercycler® Pro (Eppendorf, Germany) under the conditions of 50 °C for 30 min and 94 °C for 2 min, followed by 40 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s; finally, the last cycle was at 72 °C for 7 min followed by a final holding at 4 °C. Aliquots of the PCR amplicons were analyzed by electrophoresis on 2 % agarose gels, stained with ethidium bromide, and visualized with Quantity One Quantification Analysis Software version 4.5.2 (Gel Doc EQ System; Bio-Rad, Hercules, CA).
4
Leishmania Parasite Identification Protocol
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Amplification was performed in a PCR Mastercycler® pro (Eppendorf, Germany) with conditions as follows; denaturation at 94 °C for 4 min, followed by 40 cycles of 94 °C for 1 min’; 65 °C for 1 min; and 72 °C for 1 min, with the final extension at 72 °C for 7 min. The forward and reverse ITS1 regions of the rRNA of Leishmania parasite primers were LeF: 5′ TCC GCC CGA AAG TTC ACC GAT A 3’ and LeR: 5′ CCA AGT CAT CCA TCG CGA CAC G 3’, respectively [29 (link)]. In order to maintain that the template DNA had been extracted properly, primers that anneal to human DNA (UNFOR403: 5’-TGA GGA CAA ATA TCA TTC TGA GG-3’ and UNREV1025: 5’-GGT TGT CCT CCA ATT CAT GTT A-3’) were used [30 (link)]. Therefore, clinical samples which contain human DNA should show the PCR products of 628 bps. The products were analyzed on 1.5 % agarose gel electrophoresis, stained with 0.5 μg/ml ethidium bromide and visualized with Quantity One quantification analysis software, version 4.5.2 Gel Doc EQ system (Bio-Rad, USA). DNA from cultured Leishmania promastigotes isolated from a patient [5 (link)] was used as the positive control. DNA from saliva and EDTA blood from a healthy individual who had never traveled into endemic areas were used as negative controls.
5
Transcriptional Profiling of Fermentation Genes
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Expression of genes
NUG1, LSM8, PDC5, NSR1, GPD1 and
GUT2 was investigated by qPCR. Normalization of gene expression was carried out using
ACT1 and
RDN18-1 as controls since their expression remains constant along fermentation. Primers were designed using
Primer-BLAST (NCBI) and
S. kudriavzevii and
S. cerevisiae gene sequences were deposited in databases (
www.ncbi.nlm.nih.gov ; GSE90793). Forward and reverse oligonucleotides were synthetized to hybridize to the selected alleles of
S. kudriavzevii or
S. cerevisiae genes (
Table 1 ). PCR Mastercycler pro (Eppendorf, Germany) confirmed the allele primer specificity and their annealing temperature. cDNA synthesis and RNA extraction were carried out as previously explained. qPCR runs were done in triplicate in a LightCycler® 480 Real-Time PCR System (Roche, Switzerland) and analyzed using the software from the manufacturer (LightCycler® Software, version 4.0). The relative gene expression was quantified by comparison with
ACT1 and
RDN18-1 expression, after confirm comparable PCR efficiency.
NUG1, LSM8, PDC5, NSR1, GPD1 and
GUT2 was investigated by qPCR. Normalization of gene expression was carried out using
ACT1 and
RDN18-1 as controls since their expression remains constant along fermentation. Primers were designed using
S. kudriavzevii and
S. cerevisiae gene sequences were deposited in databases (
S. kudriavzevii or
S. cerevisiae genes (
ACT1 and
RDN18-1 expression, after confirm comparable PCR efficiency.
6
In Situ Detection of Breast Cancer Cells
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For in situ diagnosis, breast cancer cell lines were used. MCF-7, SK-BR-3, MDA-MB-231, MDA-MB-453, and HCC-1937 were purchased from the Korean Cell Line Bank (KCLB). The afc-probe was applied to each cell line for 2 hours. The afc-probe-treated cells were harvested and washed with PBS three times to remove the remaining free afc-DNA probe. Stepwise thermal decrement (−1 °C/min) from 37 °C to 4 °C was used to treat the cells in a PCR Mastercycler® pro from Eppendorf (Westbury, NY, USA). Flow cytometry with a MACSQuant VYB from Miltenyi Biotec (Auburn, CA, USA) was used to measure the fluorescent signals.
7
Saliva DNA extraction and PCR amplification
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2 mL of saliva was collected from each participant and was stored at -20°C [78 (link)]. DNA extraction was accomplished using prepIT-L2P and following the manufacturer’s recommendations (DNA Genotek inc., Ottawa, Canada). DNA was then purified using Genomic DNA Clean & Concentrator kit (Zymo Research, Irvine, CA) and quantified using SYBR Green I dye (Lonza, Walkersville, MD). Genomic DNA was diluted to 5 ng/μL prior to polymerase chain reaction (PCR). In a final volume of 20 μl, 1 μl DNA was amplified, and each reaction consisted of 0.5 μM forward primer, 0.5 μM reverse primer, 0.5 μl Taq polymerase solution, 5 mM MgCl2, 2 mM dNTPs, and 10% glycerol. Both genes were run on an Eppendorf PCR Mastercycler pro (model no. 6321, Hamburg,Germany) using a touchdown PCR cycle protocol adopted from Anchordoquy et al., [79 (link)] and modified to have a 65°C annealing temperature for 10 cycles, followed by a 55°C annealing temperature for 35 cycles.
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