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Benchtop 1kb dna ladder

Manufactured by Promega
Sourced in United States

The BenchTop 1kb DNA Ladder is a molecular weight marker used to determine the size of DNA fragments in gel electrophoresis. It contains a set of DNA fragments ranging from 250 to 10,000 base pairs, allowing for accurate sizing of samples within this range.

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3 protocols using benchtop 1kb dna ladder

1

Amplification and Extraction of Plasmids

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Plasmids were transformed and amplified in DH5α bacterial cells and grown overnight. DNA was extracted by Nucleobond Endotoxin-free Maxiprep (Takara Bio, Mountain View, CA, USA). Primers were designed using SnapGeneTM for different homology arm lengths (100, 200, 300, and 400 bp) for insertion in the TRAC locus (Table S1). Rules used for primer design were: ±50% CG content, less than 22 bp in length, and melting temperatures below 60 °C. The dsDNA was generated by PCR amplification using CloneAmp HiFi Taq polymerase (Takara Bio, Mountain View, CA, USA), forward and reverse primer (0.5 µM), plasmid DNA (15–20 ng), and nuclease-free water, in a final volume of 50 µL. Reactions were run on the ProFlexTM PCR System (ThermoFisher, Waltham, MA, USA) according to the following program: Initial denaturation at 98 °C for 30 sec, 20 cycles of each 3 steps (denaturation at 98 °C for 10 sec, annealing at +3 °C of lower melting temperature of primer for 15 sec, and extension at 72 °C for variable time based on PCR product size—5 sec/kb), final extension at 72 °C for 3 min. Two PCR reactions combined were run on 1% agarose gel for band size confirmation. The BenchTop 1kb DNA Ladder (Promega, Madison, WI, USA) was used in all experiments. To generate highly concentrated dsDNA, 8 PCR reactions were run in total and 2 of these reactions were combined in one gel slot.
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2

Dendrimer-DNA Complexation Assay

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Dendrimers/DNA complexes
at different N/P ratios (5, 15, and 30) were prepared following complexation
instructions of the transfection protocol. 250 ng of pGFP or model
DNA (herring sperm for proof-of-concept assays), both from Promega,
was used to form complexes with a proper dilution of the dendrimer
in deionized water. Complexes were then diluted to 20 μL final
volume, and 4 μL of loading dye (blue/orange loading dye, 6×
from Promega) was added to dendriplexes. 24 μL of each sample
was loaded on 0.8% (w/v) agarose gel. DNA ladder (BenchTop 1 kb DNA
Ladder, from Promega) was loaded in the first lane, while free DNA
was loaded in the last one. Electrophoresis running was performed
in Tris-acetate-EDTA buffer (1× TAE) at 150 V for 30 min. The
gel was subsequently placed into a plastic tray and incubated with
staining solution (Diamond Nucleic Acid Dye Promega diluted in 1×
TAE buffer) following the manufacturer’s instructions, for
20 min under gentle shaking, protected from light. The gel was analyzed
under UV Transilluminator BIO-RAD ChemiDoc XRS+ using a proper filter.
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3

RFLP-based Genotypic Classification of PsHV

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The DNA samples extracted from the PsHV isolates and from the reference virus (KS 144/79) were analyzed by RFLP for genotypic classification using the restriction enzyme PstI (20,000 U/mL, New England BioLabs, Inc., Ipswich, MA, USA), as recommended by the manufacturer.
The resulting DNA fragments were separated by electrophoresis on a 0.5% agarose gel in 1× Tris–acetate–EDTA buffer (Sigma–Aldrich, St. Louis, MO, USA) containing 5 μL of ethidium bromide (10 mg/mL) (Life Technologies, Grand Island, NE, USA) at room temperature under constant voltage of 20 V for about 15 h. The BenchTop 1 kb DNA Ladder (Promega, Madison, WI, USA) was used as a DNA size marker. The gels were observed at 254 nm using a MacroVue UV transilluminator (GE Healthcare, Cleveland, OH, USA) and photographed.
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