Computationally-predicted conserved regions were amplified using the Taq PCR Kit (New England Biolabs, MA) following the routine Taq PCR reaction protocol. Primers used were summarized in Table 1 . Mouse genomic DNA (Swiss Webster strain) was extracted from an adult mouse tail and used as the PCR template for all primers. A random extension sequence (CGATATAT) and the SpeI recognition sequence (ACTAGT) was added to the 5′ end of the forward primer, plus a random extension sequence and FseI recognition sequence (GGCCGGCC) was added to the 5′ end of the reverse primer. Then, the sticky end inserts were digested, gel purified, and ligated into the βGP-GFP backbone which was linearized with FseI and SpeI to generate experimental constructs (Fig. S1 ).
Taq pcr kit
Manufactured by New England Biolabs
Sourced in United States
The Taq PCR Kit is a collection of reagents for performing polymerase chain reaction (PCR) experiments. It includes the Taq DNA polymerase enzyme, necessary buffers, and other components required to amplify DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Minelute pcr purification kit, by Qiagen (2 mentions)
Mastercycler ep realplex4 s, by Eppendorf (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
λ dna, by New England Biolabs (1 mentions)
Biotin 11 dutp, by Jena Biosciences (1 mentions)
Lightcycler 96 real time pcr system, by Roche (1 mentions)
M mulv reverse transcriptase kit, by New England Biolabs (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Bsai hfv2, by New England Biolabs (1 mentions)
Plasmid miniprep kit, by New England Biolabs (1 mentions)
Flashgel system, by Lonza (1 mentions)
Miniprep kit, by Qiagen (1 mentions)
Minielute pcr purification kit, by Qiagen (1 mentions)
Agarose mp, by Roche (1 mentions)
100 bp dna ladder, by New England Biolabs (1 mentions)
Qiaquick pcr kit, by Qiagen (1 mentions)
Bigdye terminator v3, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
14 protocols using taq pcr kit
1
PCR Amplification and Cloning
2
Quantifying Biocide Impact on Bacterial DNA
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PCR was used to determine the effect of biocide treatment on DNA by amplifying 16S rDNA, LIF, and Klebsiella pneumoniae carbapenemase (KPC) target sequences. To amplify 16S sequences from eDNA samples, 25 µL PCR reaction mixtures (Taq PCR Kit, New England Biolabs, Ispwich, MA, USA) with 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) primers were used [82 (link)]. Forward (5′-ATGAAGGTCTTGGCGGCAGG-3′) and reverse (5′-ACCTCCTGCTAGAAGGCCTG-3′) primers were used to amplify the LIF gene from pC1-L samples. PCR conditions involved an initial stage of 5 min at 95 °C. This was followed by 30 cycles at 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s and a final stage at 72 °C for 5 min. Purified genomic DNA was used as a positive control and the PCR products visualized by gel electrophoresis. KPC sequences were amplified using the AmpliSense MDR KPC/OXA-48-FL reagent kit (AmpliSense, Moscow, Russia) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).
3
Synthesis of Labeled DNA Handles
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Biotin and digoxigenin labelled DNA (so-called ‘DNA handles’) were synthesized by incorporation of modified dUTPs using Taq polymerase (TaqPCR kit, NEB). The PCR mixture was setup according to the manufacturer's instructions. For synthesizing biotin labelled DNA, biotin-11-dUTP (Jena Bioscience) was added to the PCR mixture at a ratio of 1:8 of biotin-dUTP:dTTP. For synthesizing digoxigenin labelled DNA, dig-11-dUTP (Jena Bioscience) was added to the PCR mixture at a ratio of 1:8 of dig-dUTP:dTTP. The PCR for handle synthesis amplified an 860 bp section of λ-DNA (NEB). Unlabelled PCR sections were synthesized using a Taq based master mix (LongAmp Hot Start Master Mix, NEB). After the PCR amplification, samples were purified by a spin column (Qiaquick, Qiagen) according to the manufacturer's instructions. The primers used for each structure described in this study are given in the SI (Supplementary Tables S1–S8 ). The PCR protocol was optimized so that a single strong band was observed by gel analysis (see Supplementary Figure S1 ) and therefore gel purification was not needed before the golden gate assembly step.
4
Amplification of λ xis Gene Fragment
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Taq PCR kit was purchased from New England Biolabs (Ipswitch, MA, USA). The forward and reverse primers for the amplification of λ xis gene fragment were synthesized by Sigma-Aldrich and their sequences were 5'-CCTGCTCTGCCGCTTCACGC-3' and 5'-TCCGGATAAAAACGTCGATGACATTTGC-3' , respectively. The volume of the reaction mixture was 25 μL, which was composed of 2.5 μL of 10× standard Taq reaction buffer, 0.5 μL of 1 mM deoxynucleotide solution, 0.5 μL of each of the primers (10 μM), 0.125 μL of Taq DNA polymerase; selected volume of water or drugs diluted with water (sterile, ACS purity, Sigma-Aldrich) and 0.5 μL of bacteriophage λ DNA.
The PCR was performed using Mastercycler ep realplex4 S (Eppendorf, AG, Hamburg, Germany) with the following cycling conditions: initial denaturation for 120 s at 95°C; 30 cycles of PCR (15 s at 95°C, 15 s at 64°C and 45 s at 72°C) with a final elongation for 5 min at 72°C. The obtained DNA fragments (498 bp) were purified by MinElute PCR Purification Kit (Qiagen, Germany). The DNA concentration was determined spectrophotometrically.
The PCR was performed using Mastercycler ep realplex4 S (Eppendorf, AG, Hamburg, Germany) with the following cycling conditions: initial denaturation for 120 s at 95°C; 30 cycles of PCR (15 s at 95°C, 15 s at 64°C and 45 s at 72°C) with a final elongation for 5 min at 72°C. The obtained DNA fragments (498 bp) were purified by MinElute PCR Purification Kit (Qiagen, Germany). The DNA concentration was determined spectrophotometrically.
5
Quantitative Analysis of Gene Expression
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Polymerase chain reaction (PCR) and real-time PCR were performed using primers after reverse-transcription of total RNAs, as previously described (Heo et al., 2014 (link)) PCR was performed using Taq PCR Kit (New England Biolabs Ltd., Ipswich, MA). The cDNA was denatured at 90°C for 5 min followed by 25 cycles of PCR (95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec). In the analysis, transcript of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control. Real-time quantitative PCR was performed in triplicate using the LightCycler 96 Real-Time PCR System (Roche, Germany); each 20-μl reaction consisted of 10 μl of SYBR Green Master Mix, 2 μl of forward and reverse primers (10 pM each) of genes to be analyzed, and cDNA template. Thermal cycling conditions were as follows: 95°C for 10 min, and 45 cycles at 95°C for 10 sec, 50°C for 10 sec, and an elongation period for 10 sec at 72°C. The relative expression of each gene was then calculated as a ratio to GAPDH using LightCycler 96 software (Version 1.1.0.1320). Primers for glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were 5-GAGTCAACGG ATTTGGTCCT-3 (forward) and 5-TGTGGTCATGAGTCCTTCCA-3 (reverse). Primers for IL8 were 5-TCTGCAGCTCTGTGTGAAGG-3 (forward) and 5-AATTTCTGTGTTGGCGCAGT-3 (reverse).
6
Two-Step RT-PCR for Amplifying SUMO Variants
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A two-step RT-PCR was used during the initial validation of the primers designed to amplify the different SUMO variants described in this manuscript and to clone the amplified PCR products. cDNA synthesis was performed using the M-MuLV® Reverse Transcriptase kit (New England BioLabs, Inc, Ipswich, MA) according to the manufacturer’s recommendations. The reaction mix was incubated at 42 °C for 1 h and subsequently cooled down to 4 °C. Negative control samples were produced using all the ingredients minus the M-MuLV Reverse Transcriptase; nuclease-free milli-Q water was used in place of the enzyme to keep final volumes equal. The cDNA synthesized was stored in aliquots at − 80 °C. The subsequent PCR reactions were performed using the Taq PCR kit from NEB (New England BioLabs, Inc.), using 2 μL from the RT reaction as template. The resulting PCR products were ethanol precipitated and sequenced using the Sanger method at the Genomic Analysis Core Facility, Border Biomedical Research Center, at The University of Texas at El Paso. Aliquots of the PCR products obtained were also analyzed by agarose gel electrophoresis using 1.5% agarose gels in 1 × TAE buffer (40 mM Tris, 20 mM Acetate, 1 mM EDTA, pH 8.6), and used for cloning into the pJET1.2 plasmid as described below.
7
Detailed Molecular Biology Reagents
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Unless otherwise stated, all chemicals and medium compounds were bought from Sigma-Aldrich (St Louis, USA), Carbosynth (Compton, Berkshire, UK), or SERVA Electrophoresis Gmb (Heidelberg, Germany). Antibiotics were purchased from Cayman Chemical Company (Ellsworth, USA). Restriction enzymes (BsaI-HFv2, BbsI-HF), T4 DNA ligase, Taq PCR Kit, Plasmid Miniprep Kit, and all the rest necessary molecular biology reagents were bought from New England Biolabs Inc. (Ipswich, USA). Biochanin A was purchased from Carbosynth (Compton, Berkshire, UK). The UHPLC grade solvents used in this study were bought from Merck KGaA (Darmstadt, Germany).
8
Genotyping Floxed ROCK1 Alleles
9
PCR Amplification and Cloning
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Computationally-predicted conserved regions were amplified using the Taq PCR Kit (New England Biolabs, MA) following the routine Taq PCR reaction protocol. Primers used were summarized in Table 1 . Mouse genomic DNA (Swiss Webster strain) was extracted from an adult mouse tail and used as the PCR template for all primers. A random extension sequence (CGATATAT) and the SpeI recognition sequence (ACTAGT) was added to the 5′ end of the forward primer, plus a random extension sequence and FseI recognition sequence (GGCCGGCC) was added to the 5′ end of the reverse primer. Then, the sticky end inserts were digested, gel purified, and ligated into the βGP-GFP backbone which was linearized with FseI and SpeI to generate experimental constructs (Fig. S1 ).
10
Plasmid Purification and E6-HPV16 Amplification
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The plasmid was purified using the Qiagen Miniprep Kit (Qiagen, Germantown, MD, USA) and the PCR amplification of the gene was performed using a set of primers flanking the complete open reading frame: 5′-ATGCACCAAAAGAGAACTGC-3′ (E6 forward) and 5′-TTACAGCTGGGTTTCTCTAC-3′ (E6 reverse). The PCR mixture (Taq PCR kit, New England Biolabs, Ispwich, MA, USA), contained the PCR buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl with 1.5 mM MgCl2 included), 0.2 mM of dNTPs and 0.4 µM of each primers with E6-HPV16-pUC57 synthetic plasmid using as a template. The DNA amplification was carried out for 40 cycles of the denaturation at 94 °C for 30 s, the annealing at 56 °C for 30 s and the primer extension at 72 °C for 30 s. The amplified E6-HPV16 oncogene was purified using MiniElute PCR Purification Kit (Qiagen, Germantown, MD, USA). The amplified product of 477 base pairs was analyzed by agarose gel electrophoresis and the conditions were as follows: 1% agarose gel (Agarose MP, Roche Diagnostics, Indianapolis, IN, USA) in TAE buffer, at 60 V for 160 min (Bio-Rad, Hercules, CA, USA). The 100 bp DNA ladder (New England Biolabs, Ispwich, MA, USA) was used as a molecule size marker. The ethidium bromide-labeled bands were visualized via UV transilluminator at 312 nm (Vilber-Lourmat, Marne-la-Valle’e Cedex, France).
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