Q5000 micro volume uv vis spectrophotometer

The resulting PCR amplicons were diluted up to 10 ng/μL and then used as templates within the second-step PCR for further amplification, and to include the indexes (barcodes) as well as the Illumina adaptors. The combinatorial use of index primers resulted in unique samples that were pooled and sequenced on one Illumina MiSeq run. In more detail, amplification reaction was performed using the KAPA HiFi HotStart PCR Kit in a final volume of 50 μL. Each reaction contained 10 μL of KAPA HiFi Fidelity Buffer (5×), 1.5 μL of dNTPs solution (10 mM each), 5 μL of the forward indexing primer (10 μM), 5 μL of the reverse indexing primer (10 μΜ), 1 μL of KAPA HiFi HotStart DNA Polymerase (1 U/μL), 2 μL from the diluted PCR product (10 ng/μL) and 25.5 μL of sterile deionized water. The PCR amplifications were performed with a 3-min incubation at 95 °C followed by 8 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, and a final 5-min terminator reaction at 72 °C. The resulting amplicons from indexing PCR were purified using Macherey-Nagel’s NucleoMag^® NGS Clean-up and Size Selection kit according to the manufacturer’s recommendations. Amplicons from different samples were quantified with a Quawell Q5000 micro-volume UV−Vis spectrophotometer and merged in equimolar ratios (8 nM).

The resulting PCR amplicons were diluted up to 10 ng/µL and then used as templates within the second-step PCR for further amplification, and to include the indexes (barcodes) as well as the Illumina adaptors. The combinatorial use of index primers resulted in unique samples that were pooled and sequenced by Macrogen using a 2 × 300 bp pair-end kit on a MiSeq platform.
In more detail, the amplification reaction was performed using the KAPA HiFi HotStart PCR kit in a final volume of 50 µL. Each reaction contained 10 µL of KAPA HiFi Fidelity buffer (5×), 1.5 µL of dNTPs solution (10 mM each), 5 µL of the forward indexing primer (10 µM), 5 µL of the reverse indexing primer (10 µM), 1 µL of KAPA HiFi Hot Start DNA polymerase (1 U/µL), 2 µL from the diluted PCR product (10 ng/µL), and 25.5 µL of sterile deionized water. The PCR amplifications were performed with a 3-min incubation at 95 °C followed by eight cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final 5-min terminator reaction at 72 °C. The resulting amplicons from indexing PCR were purified using the NucleoMag NGS Clean-up and Size Selection kit (Macherey-Nagel, Deuren, Germany) according to the manufacturer’s recommendations. Amplicons from different samples were quantified with a Quawell Q5000 micro-volume UV–Vis spectrophotometer and merged in equimolar ratios (8 nM).

The resulting PCR amplicons were diluted up to 10 ng/μL and then used as templates within the second-step PCR for further amplification, and to include the indexes (barcodes) as well as the Illumina adaptors.
In more detail, the amplification reaction was performed using the KAPA HiFi HotStart PCR kit in a final volume of 50 μL. Each reaction contained 10 μL of KAPA HiFi Fidelity buffer (5×), 1.5 μL of dNTPs solution (10 mM each), 5 μL of the forward indexing primer (10 µ), 5 μL of the reverse indexing primer (10 µM), 1 μL of KAPA HiFi Hot Start DNA polymerase (1 U/μL), 2 μL from the diluted PCR product (10 ng/μL), and 25.5 μL of sterile deionized water. The PCR amplifications were performed with a 3 min incubation at 95 °C followed by eight cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final 5 min terminator reaction at 72 °C. The resulting amplicons from the indexing PCR were purified using the NucleoMag NGS Clean-up and Size Selection kit (Macherey-Nagel, Deuren, Germany) according to the manufacturer’s recommendations. Amplicons from different samples were quantified with a Quawell Q5000 micro-volume UV–Vis spectrophotometer and merged in equimolar ratios (8 nM). The combinatorial use of index primers resulted in unique samples that were sequenced by Macrogen using a 2 × 300 bp pair-end kit on a MiSeq platform.

Throughout the experimental procedure, imaging of the desired
amplification products was performed in a Gel Doc™ XR+ system (Bio-Rad) after
loading 5 μl from each PCR reaction on 1.5% (w/v) agarose gels and separating
them by electrophoresis. Purification of the PCR products was carried out with a
20% PEG, 2.5 M NaCl solution as previously described [113 (link)]. The concentration of purified PCR
product was measured with a Quawell Q5000 micro-volume UV-Vis spectrophotometer.
Purified PCR products were sequenced using the appropriate primers in each case
(Additional file 2) while clonedWolbachia PCR products were sequenced
with the universal primers T7 and SP6. In this case, at least three
transconjugants were sequenced as previously described [86 (link)]. A dye terminator-labelled cycle
sequencing reaction was conducted with the BigDye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems). Reaction products were purified using an
ethanol/EDTA protocol according to the manufacturer’s instructions (Applied
Biosystems) and were analyzed in an ABI PRISM 3500 Genetic Analyzer (Applied
Biosystems).

Plants infected with BSGM were obtained from four major barley-producing areas in Morocco: Abda, Rabat-Salé-Zemmour-Zaër, Fès-Meknes, and Doukkala. All infested plant samples were kept at the International Center of Agricultural Research in Dry Areas (ICARDA) in Rabat, Morocco (Table 1), where the BSGM larvae and pupae were collected during the winter of 2020. Larvae were stored in absolute ethanol at −20 °C, whereas pupae protected in infested plant tissues were reared in a growth room under constant conditions (temperature 23 ± 2 °C, relative humidity 65 ± 5%). Once the adults emerged, they were stored in 100% ethanol at −20 °C. Before DNA extraction, each sample was surface sterilized with a 70% v/v ethanol solution, rinsed with sterile deionized water to remove traces of ethanol, left to dry on a sterile surface, and placed in 1.5 mL centrifuge tubes. After surface sterilization, whole-fly DNA was isolated using a modified CTAB (cetyl trimethyl ammonium bromide) method [36 (link)]. The quantity and quality of the DNA preparations and the concentration of double-stranded DNA were both analyzed by a Q5000 micro-volume UV-Vis spectrophotometer (Quawell Technology, San Jose, CA, USA). DNA samples were stored in Eppendorf tubes at −20 °C until further analysis.

The purified PCR products were diluted to a final concentration of 10 ng/μl and submitted to indexing PCR in order to incorporate the Illumina index primers to their sequence. During indexing PCR, each sample was amplified with a unique combination of index primers. Amplification was performed in 50 μl reactions using the KAPA HiFi HotStart PCR Kit. Each reaction contained 10 μl of KAPA HiFi Fidelity Buffer (5X), 1.5 μl of dNTPs solution (10 mM each), 5 μl of the forward index primer (10 μM), 5 μl of the reverse index primer (10 μΜ), 1 μl of KAPA HiFi HotStart DNA Polymerase (1 U/μl), 2 μl from the diluted PCR product (10 ng/μl) and 25.5 μl of sterile deionized water. The PCR program was comprised of an initial denaturation step at 95 °C for 3 min, followed by 8 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s. The reaction was terminated with a final extension step at 72 °C for 5 min. The resulting amplicons were purified using Macherey-Nagel’s NucleoMag® NGS Clean-up and Size Selection kit according to the manufacturer’s recommendations. Purified samples were suspended in 30 μl of sterile deionized water and their concentration was measured with a Quawell Q5000 micro-volume UV-Vis spectrophotometer. All samples were diluted to a final concentration of 8 nM and mixed equimolarly.