Benchtop 1kb dna ladder

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 SnapGene^TM 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 ProFlex^TM 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.

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.