Two pairs of polymorphic SSR markers (E354010801 and G6203) developed for cowpea genetic diversity analysis [8 (link)] were used to confirm the hybridity of the F1 plants. The markers were first confirmed to be polymorphic between each pair of parents, before being used to genotype the offspring of crosses. Genomic DNA samples from fresh young leaves of the parents and hybrid plants were isolated and purified using NucleoMag Plant DNA extraction kit (NucleoMag, Germany). A NanoDrop 2000 Ultraviolet–Visible Spectrophotometer (Thermo Fisher Scientific) was used to quantify and assess the quality of the extracted DNA samples. For each genotyping reaction, a 20 µL reaction volume containing 40 ng of genomic DNA, 1× KAPA Taq ReadyMix DNA Polymerase (KAPABiosystems) and 0.5 pmol/µL of forward and reverse primers was subjected to polymerase chain reaction (PCR) in a Bio-Rad thermal cycler (BIORAD, Japan). The recommended thermal cycling program for the markers [8 (link)] was adopted to amplify the SSR loci and the resulting PCR products were analyzed using a Microchip Electrophoresis System for DNA/RNA analysis (MultiNA, Shimadzu, Japan).
Nanodrop 2000 ultraviolet visible spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop 2000 Ultraviolet–Visible Spectrophotometer is a compact, benchtop instrument designed for the quantification and analysis of nucleic acids and proteins. It utilizes a patented sample retention system that allows for the measurement of sample volumes as low as 0.5 microliters, without the need for cuvettes or other sample containment devices. The NanoDrop 2000 provides accurate and reproducible results with a wide linear range and minimal sample consumption.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using nanodrop 2000 ultraviolet visible spectrophotometer
1
Confirming Hybridity of Cowpea F1 Plants
2
Amplification and Extraction of Plasmid Library
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Based on the results of the storage capacity test, 1 million clones were obtained. The required connecting liquid volume was then calculated, and the systems were transformed into the HST08 competent cells (TaKaRa, Dalian, China). The cells were coated on 10 LB ampicillin (100 μg/mL) 24.5 cm agar plates and cultured overnight at 37°C. The number of amplified clones obtained from the actual transformation was calculated. LB liquid medium (5 mL) was added to each plate and all clones were washed with a glass rod and collected into a triangular flask. The transferred clones were shaken at 225 × g and 37°C for 3 h, and the plasmids of the amplified library were extracted by using a Nucleobond Xtra MIDI EF kit. The concentration and purity of the extracted plasmid were determined by using a NanoDrop-2000 ultraviolet-visible spectrophotometer (Thermo Fisher, USA). The plasmids in 100 ng of the amplified library were detected by performing 1.5% agarose gel electrophoresis.
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