Five-hundred picomoles of reverse primer (Integrated DNA Technologies) were radiolabeled using 1 µL of 250 μCi of γ32P ATP (PerkinElmer) and 50 units of T4 polynucleotide kinase (PNK, New England Biolabs) at 37°C for 90 min in 1× T4 PNK Buffer. Following incubation, 5′ end-labeled primers were PAGE purified. A NanoDropOne spectrophotometer (Thermo Fisher Scientific) was used to determine concentration of primers prior to use in primer extension assays.
Reverse primer
Manufactured by Integrated DNA Technologies
Sourced in United States
A reverse primer is a short, synthetic DNA sequence that is designed to be complementary to the reverse strand of a target DNA sequence. It serves as a starting point for DNA synthesis during the polymerase chain reaction (PCR) process.
Automatically generated - may contain errors
Lab products found in correlation
Forward primer, by Integrated DNA Technologies (10 mentions)
Eb buffer, by Qiagen (2 mentions)
Agarose e gel, by Thermo Fisher Scientific (2 mentions)
Ultrapure water, by Thermo Fisher Scientific (2 mentions)
Phusion hs 2 polymerase, by Thermo Fisher Scientific (2 mentions)
Idt oligo analyzer, by Integrated DNA Technologies (2 mentions)
Taq dna polymerase 2x master mix red, by Ampliqon (2 mentions)
Taq dna polymerase 2 master mix red, by Ampliqon (2 mentions)
T4 polynucleotide kinase pnk, by New England Biolabs (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Taqman probe, by Integrated DNA Technologies (1 mentions)
Betaine, by Merck Group (1 mentions)
Deoxynucleotide triphosphate, by Thermo Fisher Scientific (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Evagreen, by Biotium (1 mentions)
Ultra pure pcr water, by Quality Biological (1 mentions)
Tapestation 4200, by Agilent Technologies (1 mentions)
15 protocols using reverse primer
1
Radiolabeling of Reverse Primers
2
Validating Isoform-Specific PCR Primers
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Primers flanking isoform junctions were designed using Primer3 (primer3.sourceforge.net). The thermodynamic suitability of the primer pairs was verified using the IDT Oligo Analyzer (Integrated DNA Technologies, USA) and in silico PCR was carried out using the UCSC genome browser to ensure primer specificity. Primers were synthesized by Integrated DNA Technologies (USA) and resuspended to 100 μM stock concentration with EB buffer (Qiagen, USA).
To validate the presence of the isoform junctions, a 50 μl PCR reaction was set up using primers flanking the junction under investigation. The reaction consisted of 0.5 μl of Phusion HS II polymerase (1 U; Thermo Scientific), 10 μl of 5x HF buffer, 1.5 μl of DMSO, 1 μl of 10 mM dNTPs, 0.5 ng of cDNA template, 2 μl of forward primer (10 mM; Integrated DNA Technology, USA) and 2 μl of reverse primer (10 mM; Integrated DNA Technologies, USA). Each reaction was topped up to 50 μl with Ultrapure water (Invitrogen, USA). Conditions for PCR amplification were as follows: 98 °C for 1 min then 35 cycles at 98 °C (15 s)/63 °C (15 s)/72 °C (15 s) followed by a final extension for 5 min at 72 °C. For visualization, 10 μl from each PCR reaction was diluted 1:2 with Ultrapure water, loaded onto a 1% Agarose E-gel (Invitrogen, USA) and run for 10 min.
To validate the presence of the isoform junctions, a 50 μl PCR reaction was set up using primers flanking the junction under investigation. The reaction consisted of 0.5 μl of Phusion HS II polymerase (1 U; Thermo Scientific), 10 μl of 5x HF buffer, 1.5 μl of DMSO, 1 μl of 10 mM dNTPs, 0.5 ng of cDNA template, 2 μl of forward primer (10 mM; Integrated DNA Technology, USA) and 2 μl of reverse primer (10 mM; Integrated DNA Technologies, USA). Each reaction was topped up to 50 μl with Ultrapure water (Invitrogen, USA). Conditions for PCR amplification were as follows: 98 °C for 1 min then 35 cycles at 98 °C (15 s)/63 °C (15 s)/72 °C (15 s) followed by a final extension for 5 min at 72 °C. For visualization, 10 μl from each PCR reaction was diluted 1:2 with Ultrapure water, loaded onto a 1% Agarose E-gel (Invitrogen, USA) and run for 10 min.
3
Validating Isoform-Specific Primers
4
Multiplex PCR for Uropathogenic E. coli
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Previously described PCR conditions and primers were used to amplify the genes encoding adhesins (papA, papGI, papGII, papGIII, fimH, afa, sfaS, iha, focG, and bmaE), toxins (cnf1, hlyA, tsh, usp, set 1 and astA), iron acquisition systems (iuc, iroN, irp2, feoB, fyuA, and ireA), protectins (kpsMT, ompT, iss, and traT), and pathogenicity islands (malX) [28 (link),29 (link)]. Each uniplex PCR assay was developed using 20 μL of the reaction mix that included 12 μL Taq DNA Polymerase 2X Master Mix RED (Ampliqon), 1 μL forward primer, 1 μL reverse primer (Integrated DNA Technologies), 3 μL nuclease-free water, and 3 μL DNA template (300 ng). The concentration of the primers used was 10 pmol.
5
Identification of K1 and K2 Capsular Serotypes
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The primers and PCR conditions used to identify the K1 and K2 capsular serotypes were as previously described [19 (link)]. Each separate uniplex PCR assay was performed using 20 μL of reaction mix that included: 12 μL of Taq DNA Polymerase 2X Master Mix RED (AMPLIQON, Odense, Denmark), 1 μL of forward primer and 1 μL of reverse primer (10 pmol, Integrated DNA Technologies), 3 μL of nuclease-free water, and 3 μL of DNA template (100 ng).
6
Quantification of PMMoV in Human Feces
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Quantification of PMMoV concentration (in terms of copies/L) was performed by RT-qPCR assay. PMMoV is the most abundant RNA virus in human feces. It was previously proposed as a potential viral indicator for human fecal contamination due to its ubiquitous global distribution without substantial seasonal fluctuations (Kitajima et al., 2018 ). Quantitative PCR for PMMoV was performed using forward primer (5′- GAGTGGTTTGACCTTAACGTTTGA-3′), reverse primer (5′-TTGTCGGTTGCAATGCAAGT-3′), and Taqman probe (5′-FAM-CCTACCGAAGCAAATG-ZEN/IBFQ-3′) (Integrated DNA Technologies, Leuven, Belgium). Gblock for PMMoV is listed in Table S3. A thermal cycler profile of 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s and 55 °C for 30 s was used (Haramoto et al., 2013 (link)). For PMMoV qPCR, a LOD of 10 copies/well and an amplification efficiency of 95.7 % was achieved.
7
Isothermal Amplification Assay for Rapid Detection
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The iso-IMRS amplification
assay uses one forward and one reverse primer, which bind to 52 and
55 sites, respectively. The 25 μL reaction mixture uses 640
U/μL Bst 2.0 polymerase (New England Biosciences),
with 1× isothermal amplification buffer, 3.2 mM forward primer,
and 1.6 mM reverse primer (Integrated DNA Technologies) combined with
10 mM dNTPs (Agilent), 0.4 M Betaine (Sigma-Aldrich), and 2% molecular-biology-grade
Ficoll-400 (Sigma-Aldrich). The sample (5 μL) is included in
each 25 μL reaction. For real-time amplification reactions,
0.2× EvaGreen intercalating dye and 30 mM ROX reference dye are
included. Amplification is carried out at 56 °C for 40 min, followed
by a melting curve analysis for real-time amplification. Amplified
products were visualized by gel electrophoresis on 10% acrylamide
gels.
assay uses one forward and one reverse primer, which bind to 52 and
55 sites, respectively. The 25 μL reaction mixture uses 640
U/μL Bst 2.0 polymerase (New England Biosciences),
with 1× isothermal amplification buffer, 3.2 mM forward primer,
and 1.6 mM reverse primer (Integrated DNA Technologies) combined with
10 mM dNTPs (Agilent), 0.4 M Betaine (Sigma-Aldrich), and 2% molecular-biology-grade
Ficoll-400 (Sigma-Aldrich). The sample (5 μL) is included in
each 25 μL reaction. For real-time amplification reactions,
0.2× EvaGreen intercalating dye and 30 mM ROX reference dye are
included. Amplification is carried out at 56 °C for 40 min, followed
by a melting curve analysis for real-time amplification. Amplified
products were visualized by gel electrophoresis on 10% acrylamide
gels.
8
dPCR Assay for Environmental Monitoring
9
Bacterial 16S rRNA Gene Sequencing
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The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal bacterial primers. Forward Primer (5′ GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C 3′)(Integrated DNA Technologies) and Reverse Primer (5′ TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG 3′) (4 nmol Ultramer® DNA Oligo 55 bases : Integrated DNA Technologies)47 (link). Library preparation was performed according to the standard instructions of the 16S Metagenomic Sequencing Library Preparation protocol (IlluminaTM, Inc., San Diego, CA, United States). The size of the amplicons were then visualised using the 4200 TapeStation (Agilent Technologies). Two negative controls were used in the amplicon preparation step. The extraction negative control as well as no template control were used in the initial amplification step. No amplification was observed in either of the negative controls.
Libraries were then sequenced on the MiSeq platform using MiSeq Reagent kit v3 (Illumina) and paired-end 2 × 300 bp sequencing was performed at the Sequencing Core Facility, National Institute for Communicable Diseases, South Africa.
Libraries were then sequenced on the MiSeq platform using MiSeq Reagent kit v3 (Illumina) and paired-end 2 × 300 bp sequencing was performed at the Sequencing Core Facility, National Institute for Communicable Diseases, South Africa.
10
Antibiotic Resistance Gene Detection in K. pneumoniae
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The conventional polymerase chain reaction (PCR) method was used to determine on the chromosome of the K. pneumoniae strains; the presence or absence of the genes encode resistance to beta–lactams (blaSHV, blaCITM, blaTEM, blaCTX-1, blaCTXM-2, and blaCTXM-9), streptomycin (aadA1), gentamicin (aac(3)-IV), sulfonamide (sul1), chloramphenicol (cat1 and cmlA), tetracycline (tet(A) and tet(B)), trimethoprim (dfrA1), and quinolones (qnr). The primers and PCR conditions used for detection of antibiotic resistance genes were as described previously [48 (link),49 (link)]. The final volume-per-reaction mixture for each uniplex PCR assay was 20 µL; 1 µL of forward primer and 1 µL of reverse primer (10 pmol, Integrated DNA Technologies, San Diego, CA, USA), 12 µL of Taq DNA Polymerase 2× Master Mix RED (AMPLIQON, Copenhagen, Denmark), 3 µL of nuclease- free water, and 3 µL of DNA template (100 ng). The K. pneumoniae strains from clinical isolates Kp7, Kp17, Kp37, Kp39, and Kp74, which harbor the antibiotic-resistance genes studied, were used as positive controls.
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