At least two cysticerci obtained previously from each carcass were macerated individually, and the parasite’s DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen®, Cat. No. 69504, Hilden, Germany), according to the manufacturer’s instructions. To differentiate the species and genotypes of Taenia spp., a multiplex PCR was used [22 (link)], based on the nucleotide sequences of Cox1 [Table 1 ]. A volume of 25 µL of reaction mixture was used for amplification with the Hot Start Taq Master Mix kit (Qiagen®, Cat. No.203446). Electrophoresis was performed on the PCR products in 1% agarose gel for 55 min at 90 V. Positive controls were donated by the Institute of Parasitology Justus Liebig University, Giessen. Sequence of specific primers and the conditions used in the multiplex PCR are described in Table 1 .
Hot start taq master mix kit
Manufactured by Qiagen
Sourced in United States, Germany
The Hot Start Taq Master Mix kit is a pre-mixed solution containing all the necessary components for performing polymerase chain reaction (PCR) amplification, including the Taq DNA polymerase enzyme, dNTPs, and buffer. The kit is designed to provide a convenient and reliable way to perform PCR reactions, with the added benefit of a 'hot start' mechanism to prevent non-specific amplification.
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Lab products found in correlation
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Ssofast evagreen supermix, by Bio-Rad (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Quick ligation kit, by New England Biolabs (1 mentions)
Alui restriction enzyme, by Promega (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Rotor gene q, by Qiagen (1 mentions)
Biosprint 96 one for all vet kit, by Qiagen (1 mentions)
Molecular biology grade water, by Merck Group (1 mentions)
Mx3005p rt pcr platform, by Agilent Technologies (1 mentions)
8 protocols using hot start taq master mix kit
1
Molecular Identification of Taenia spp.
2
RT-PCR and Real-time PCR Protocol
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Total RNA from cells was extracted by using the RNeasy Mini kit, following manufacturer's instructions, and reverse-transcribed by the QuantiTect Reverse Transcription kit (Qiagen, Valencia, CA, USA). RT-PCRs were performed by the HotStartTaq Master Mix kit (Qiagen, Valencia, CA, USA). Primer sequences are listed in Table 2 . Thermal cycle conditions were as follows: 95°C for 15 min, 35 cycles of denaturation at 95°C for 1 min followed by annealing and extension at 72°C for 1 and 10 min, respectively. Detection of the RT-PCR products was performed by agarose gel electrophoresis and ethidium bromide staining.
Real-time PCR was performed with SsoFast Eva Green Supermix (Ref. 172-5201, Bio-Rad, CA, USA). Samples were amplified using the following thermal profile: 95°C for 30 s, 40 cycles of denaturation at 95°C 15 s followed by annealing for 30 s and 72°C for 30 s, with a final step at 65°C for 5 s. GAPDH and β-actin were used as housekeeping genes.
Real-time PCR was performed with SsoFast Eva Green Supermix (Ref. 172-5201, Bio-Rad, CA, USA). Samples were amplified using the following thermal profile: 95°C for 30 s, 40 cycles of denaturation at 95°C 15 s followed by annealing for 30 s and 72°C for 30 s, with a final step at 65°C for 5 s. GAPDH and β-actin were used as housekeeping genes.
3
Linker PCR for Random Insertion Validation
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Linker PCR was used to test individual transformant colonies and to confirm individual random-insertion events. DNA (2.5 μg) was digested with the AluI restriction enzyme (Promega) and purified by using a MinElute PCR purification kit (Qiagen). A linker, formed by annealing of oligonucleotides 254 (5′-CGACTGGACCTGGA-3′) and 256 (5′-GATAAGCAGGGATCGGAACCTCCAGGTCCAGTCG-3′), was ligated to purified fragments (50 ng) with a Quick ligation kit (NEB). Linker PCR was performed with linker- and transposon-specific oligonucleotides (258 [5′-GATAAGCAGGGATCGGAACC-3′] and 5′-GCAATGTAACATCAGAGATTTTGAG-3′, respectively) by using a HotStart Taq Mastermix kit (Qiagen) and thermocycling conditions of 95°C for 5 min; 35 cycles of 94°C for 45 s, 56°C for 1 min, and 72°C for 1 min; and 72°C for 10 min. The resulting amplicons were separated on 1.5% agarose gels at 100 V for 60 min.
4
Genomic DNA Extraction and HPV Genotyping
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Genomic DNA was prepared using a Qiagen DNeasy blood and tissue kit, with the added step of incubating the paraffin sections at 120°C for 20 min in the ATL buffer before digestion with proteinase K. A reagent blank extraction control (an extraction without formalin fixed paraffin embedded – FFPE tissues) was performed during the DNA extraction procedure. The gDNA extracts were quantified with a NanoDrop spectrophotometer (Thermo Scientific). The DNA quality was tested by the amplification of a 268-bp fragment of the β-globin gene using primers G073 (5’-GAAGAGCCAAGGACAGGTAC-3’) and G074 (5’-CAACTTCATCCACGTTCACC-3’). The HPV genotypes were identified by BLAST via the US National Center for Biotechnology Information.
Negative controls [no DNA (water) and a reagent blank] were used in parallel with all PCR analyses. Positive control (HPV18) was DNA extracted from HeLa cells.
Cycling conditions using Hot Start Taq Master Mix kit from QIAGEN: 95°C 15 min 1 cycle followed by 30 s 95°C, 30 s 55°C, 30 s, and 72°C 35 cycles.
Human papilloma virus identification was considered positive if sequences were identified at least twice in DNA extracted from the same specimens.
Negative controls [no DNA (water) and a reagent blank] were used in parallel with all PCR analyses. Positive control (HPV18) was DNA extracted from HeLa cells.
Cycling conditions using Hot Start Taq Master Mix kit from QIAGEN: 95°C 15 min 1 cycle followed by 30 s 95°C, 30 s 55°C, 30 s, and 72°C 35 cycles.
Human papilloma virus identification was considered positive if sequences were identified at least twice in DNA extracted from the same specimens.
5
Cryptosporidium Detection via qPCR
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DNA amplification was performed in a Rotor-Gene Q instrument (QIAGEN GmbH, Hilden-Germany) in 25 μL reactions using the HotStart Taq master mix kit (QIAGEN, Germany), 3.5 mM MgCl2, 500 nM forward primer (crypto-F) 5’- CGC TTC TCT AGC CTT TCA TGA -3’, 500 nM reverse primer (crypto-R) 5’- CTT CAC GTG TGT TTG CCA AT-3’, 175 nM Crypto probe 5’-ROX-CCA ATC ACA GAA TCA TCA GAA TCG ACT GGT ATC–BHQ2-3’. The primer and probe sequences and corresponding assession numbers have been published elsewhere [20 (link)]. These primers and probe sequencies are specific for Cryptosporidium parvum and C. hominis, although their efficiency at detecting other Cryptosporidium species has not be evaluated.
All samples were tested in duplicate for Cryptosporidium species detection employing the following cycling protocol: one cycle at 95°C for 15 min (polymerase activation), followed by 45 cycles of 95°C for 15 seconds (denaturation), 67°C for 30 seconds (annealing) and 72°C for 30 seconds (extension), followed by a final cooling step at 40°C for 30 seconds. Phocin Herpes Virus (PhHV) Plasmid was incorporated into the master mix to control for PCR inhibitors and only samples with a cycle threshold < = 38 were considered positive.
All samples were tested in duplicate for Cryptosporidium species detection employing the following cycling protocol: one cycle at 95°C for 15 min (polymerase activation), followed by 45 cycles of 95°C for 15 seconds (denaturation), 67°C for 30 seconds (annealing) and 72°C for 30 seconds (extension), followed by a final cooling step at 40°C for 30 seconds. Phocin Herpes Virus (PhHV) Plasmid was incorporated into the master mix to control for PCR inhibitors and only samples with a cycle threshold < = 38 were considered positive.
6
Molecular Detection of Feline Pathogens
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Tissues samples of carcasses which resulted qPCRFeMV positive were further tested for feline infectious peritonitis virus (FIPV, Dual IPC‑TaqVetTM, LSI, Lissieu, France), feline immunodeficiency virus (FIV, gag protein gene‑genesig® Advanced Kit, Rownhams, UK) and feline leukemia virus (FeLV, U3 region LTR‑ genesig® Advanced Kit, Rownhams, UK). DNAs purified by means the BioSprint 96 One-For-All Vet Kit (Qiagen, Hilden, Germany) were tested for feline panleukopenia virus (FPLV, HotStartTaq Master Mix Kit, Qiagen, Hilden, Germany), for Leishmania spp. (Rodgers et al., 1990 (link)) and for Leptospira spp. (Stoddard et al., 2009 (link)).
7
Real-time PCR Amplification Protocol
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PCR for all methods was performed in a final volume of 15 μl, using the HotStart Taq Master Mix Kit from Qiagen (product 203445). The reactions contained 25 pmol of forward and reverse primers, each in 1 μl, 7.5 μl of kit master mix, 1.5-μl non-acetylated bovine serum albumin (BSA, 10 mg ml -1 , Sigma B4287) and 3 μl of the template. The intercalating dye EVAGreen (Biotium, code BT31000) was used to report product formation in non probe-based PCR reactions. The kit magnesium ion concentration of 1.5mM per reaction was supplemented to 2mM for PCR methods using EVAGreen dye and to 3mM MgCl2 for real-time PCR when using any of the labeled hybridization probes. All probes were used at a final concentration of 100 nM. The volumes were made up to 15 μl with molecular biology grade water (Sigma-Aldrich). After an initial activation step of 14 min at 95 0 C, 41-45 cycles of amplification were performed on an Mx3005P RT-PCR platform (Agilent Technologies). When using the GC-rich pks15/1 probe, an extra 4 th PCR step of 10s was included to acquire fluorescence data at 85C. Non-template controls contained water in place of DNA extract.
8
Extraction and RT-PCR Analysis of RNA
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Total RNA from cells was extracted by using the RNeasy ® Mini kit, following manufacturer's instructions, and reverse-transcribed by the QuantiTect ® Reverse Transcription kit (Qiagen, Valencia, CA, USA). RT-PCRs were performed by the HotStartTaq Master Mix kit (Qiagen, Valencia, CA, USA). Primer sequences are listed in Table 1. Thermal cycle conditions were as follows: 95°C for 15 min, 35 cycles of denaturation at 95°C for 1 min followed by annealing and extension at 72°C for 1 and 10 min, respectively. Detection of the RT-PCR products was performed by agarose gel electrophoresis and ethidium bromide staining.
Real-time PCR was performed with SsoFast Eva Green Supermix (Ref. 172-5201, Bio-Rad, CA, USA). Samples were amplified using the following thermal profile: 95°C for 30 s, 40 cycles of denaturation at 95°C 15 s followed by annealing for 30 s and 72°C for 30 s, with a final step at 65°C for 5 s. β-actin and GAPDH were used as housekeeping genes.
Real-time PCR was performed with SsoFast Eva Green Supermix (Ref. 172-5201, Bio-Rad, CA, USA). Samples were amplified using the following thermal profile: 95°C for 30 s, 40 cycles of denaturation at 95°C 15 s followed by annealing for 30 s and 72°C for 30 s, with a final step at 65°C for 5 s. β-actin and GAPDH were used as housekeeping genes.
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