The Promega Wizard genomic DNA purification kit (Promega Corporation, Madison, WI, USA) was used to extract the genomic DNA of E. coli bacterial cultures according to the manufacturer’s instructions. The universal forward (27F, 5′-AGAGTTTGATC[A/C]TGGCTCAG -3′) and reverse (1492R, 5′-G[C/T]TACCTTGTTACGACTT -3′) primers (70 ) were used to amplify a nearly full-length sequence of the 16S rRNA-encoding gene by PCR, using a reaction mixture (25 μl) composed of 2.5 μl 10× Taq buffer (100 mM Tris-HCl, pH 8), 1.25 mM MgCl2, 100 μM deoxynucleoside triphosphates (dNTPs) (Invitrogen, Carlsbad, CA, USA), 1.2 μM forward and reverse primers, 0.5 U Taq DNA polymerase (Invitrogen, USA), and about 5 ng template genomic DNA. A thermal cycler (model 2720; Applied Biosystems, Foster City, CA, USA) was used for the PCR amplification, which was performed with the following PCR program: 95°C for 5 min (initial denaturation) and then 35 amplification cycles of 94°C for 1 min (denaturation), 56°C for 1 min (annealing), and 72°C for 1 min (extension), followed by a final extension at 72°C for 10 min. Agarose gel electrophoresis was used for analyzing the PCR amplification products on agarose (1%) gels containing ethidium bromide (5 μg/ml) with DNA size marker (1 kb Plus DNA ladder; Invitrogen, USA).
Kb plus dna ladder
Manufactured by Thermo Fisher Scientific
Sourced in United States
The kb Plus DNA ladder is a molecular weight standard used for sizing and quantifying DNA fragments in agarose gel electrophoresis. It contains a mixture of DNA fragments of known sizes, ranging from 100 base pairs to 12,000 base pairs, which can be used as a reference to determine the size of unknown DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Wizard genomic dna purification kit, by Promega (2 mentions)
Deoxynucleoside triphosphates dntps, by Thermo Fisher Scientific (2 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (2 mentions)
Dneasy plant tissue kit, by Qiagen (1 mentions)
Toptaq dna polymerase, by Qiagen (1 mentions)
Mycycler thermal cycler, by Bio-Rad (1 mentions)
5 protocols using kb plus dna ladder
1
Genomic DNA Extraction and 16S rRNA Amplification
2
Comprehensive Pterocarya Species Phylogeny
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In this study, we used twenty-two individuals that represented all species and varieties and covered the entire range of each of the species (n: number of individuals, p: population number): P. fraxinifolia (n = 3, p = 3), P. hupehensis (n = 4, p = 4), P. macroptera var. delavayi (n = 2, p = 1), P. macroptera var. insignis (n = 1, p = 1), P. macroptera var. macroptera (n = 3, p = 3), P. rhoifolia (n = 3, p = 3), P. stenoptera (n = 3, p = 2), and P. tonkinensis (n = 3, p = 1) (Figure 6 ). Juglans mandshurica and Cyclocarya paliurus were used as outgroups. The voucher specimens are housed in the herbarium of the Shanghai Chenshan Botanical Garden (CSH), at Niigata University, and at Tarbiat Modares University (TMU). None of the field collections of Pterocarya species required specific permissions or involved endangered or threatened species.
DNA extraction was performed with a Qiagen DNeasy Plant Tissue Kit from silica-gel dried leaves according to the manufacturer’s (Qiagen, Valencia, CA, USA) standard protocol. The DNA extraction quality was checked by 1% agarose gel in conjunction with 1 KB Plus DNA Ladder (Invitrogen) or a New England Biolabs 100 bp DNA ladder marker (Ipswich, MA, USA). The genomic DNA concentrations were subsequently quantified with a dsDNA HS kit on a Qubit 2.0 Fluorometer.
DNA extraction was performed with a Qiagen DNeasy Plant Tissue Kit from silica-gel dried leaves according to the manufacturer’s (Qiagen, Valencia, CA, USA) standard protocol. The DNA extraction quality was checked by 1% agarose gel in conjunction with 1 KB Plus DNA Ladder (Invitrogen) or a New England Biolabs 100 bp DNA ladder marker (Ipswich, MA, USA). The genomic DNA concentrations were subsequently quantified with a dsDNA HS kit on a Qubit 2.0 Fluorometer.
3
Molecular Identification of A. marginale
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For A. marginale identification, two unequivocal specie-specific genes were targeted: msp5, a single copy gene that encodes the outer major surface protein MSP5 and msp1β a three-copy gene that encodes the outer major surface protein MSP1b from A. marginale. For both target genes, PCR reactions were conducted using primers previously reported [26 (link),27 (link)]. The molecular amplifications were performed in a 50 µL reaction mixture (0.4 µmol of each primer, 0.2 mM of each deoxyribonucleotide triphosphate, 1.25 U of TopTaq DNA polymerase (QIAGEN. Hilden, Germany), 5 µL of 10× PCR buffer and purified water for 50 µL of final volume) using 200 ng of genomic DNA (from both blood or tick samples). Amplifications were carried out in a thermocycler (Bio-Rad MyCycler Thermal Cycler. Hercules, CA, USA) under previously described cycling conditions. For each amplification reaction, positive (DNA from A. marginale Mercedes strain) and negative (pure water) controls were included. An aliquot of 5 µL of each amplified product was analyzed by electrophoresis in 1.5% agarose gel stained with ethidium bromide. A molecular size marker (1 Kb Plus DNA Ladder, Invitrogen. Carlsbad, CA, USA) was used to determine PCR product size.
4
Genomic DNA Extraction and 16S rRNA Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Promega Wizard genomic DNA purification kit (Promega Corporation, Madison, WI, USA) was used to extract the genomic DNA of E. coli bacterial cultures according to the manufacturer’s instructions. The universal forward (27F, 5′-AGAGTTTGATC[A/C]TGGCTCAG -3′) and reverse (1492R, 5′-G[C/T]TACCTTGTTACGACTT -3′) primers (70 ) were used to amplify a nearly full-length sequence of the 16S rRNA-encoding gene by PCR, using a reaction mixture (25 μl) composed of 2.5 μl 10× Taq buffer (100 mM Tris-HCl, pH 8), 1.25 mM MgCl2, 100 μM deoxynucleoside triphosphates (dNTPs) (Invitrogen, Carlsbad, CA, USA), 1.2 μM forward and reverse primers, 0.5 U Taq DNA polymerase (Invitrogen, USA), and about 5 ng template genomic DNA. A thermal cycler (model 2720; Applied Biosystems, Foster City, CA, USA) was used for the PCR amplification, which was performed with the following PCR program: 95°C for 5 min (initial denaturation) and then 35 amplification cycles of 94°C for 1 min (denaturation), 56°C for 1 min (annealing), and 72°C for 1 min (extension), followed by a final extension at 72°C for 10 min. Agarose gel electrophoresis was used for analyzing the PCR amplification products on agarose (1%) gels containing ethidium bromide (5 μg/ml) with DNA size marker (1 kb Plus DNA ladder; Invitrogen, USA).
5
GAPDH Gene Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to verify DNA integrity and the presence of potential inhibitors, negative samples were subjected to PCR for the detection of the GAPDH gene using primers developed by Birkenheuer et al. (2003) (link) (Table 1). In the PCR reactions, we used the final volume of 25μL, composed of 5μL of genomic DNA, 10x reaction buffer, 2.0mM MgCl 2 , 0.2mM of each dNTP, 0.4μM of each primer, 1.25U of Taq polymerase, and ultrapure water until the final volume was achieved. The amplification protocol used was the one published by Lacerda et al. (2017) (link), and consisted of a initial denaturation step at 95°C for 5 minutes followed by 40 cycles of 94°C for 30 seconds for denaturation, annealing at 52°C for 1 minute, extension at 72°C for 1 minute, and a final extension step at 72°C for 5 minutes.
PCR products were detected by 2% agarose gel electrophoresis on a TAE running buffer (40mM Tris-acetate, 2mM EDTA pH 8.0). The gel was run at 80V, 180mA for 30 minutes, and then stained with ethidium bromide (0.5μg/mL). A standard primer pair (1 Kb Plus DNA Ladder -Invitrogen ) was used to estimate the size of the amplified products. Amplified products were visualized under a ultraviolet (UV) transilluminator (LPIX, LoccusBiotecnologia ) and photographed on a coupled image analyzer.
PCR products were detected by 2% agarose gel electrophoresis on a TAE running buffer (40mM Tris-acetate, 2mM EDTA pH 8.0). The gel was run at 80V, 180mA for 30 minutes, and then stained with ethidium bromide (0.5μg/mL). A standard primer pair (1 Kb Plus DNA Ladder -Invitrogen ) was used to estimate the size of the amplified products. Amplified products were visualized under a ultraviolet (UV) transilluminator (LPIX, LoccusBiotecnologia ) and photographed on a coupled image analyzer.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!