Partial panC gene was amplified using following primer sequences for groups I to VI according to Guinebretière et al. (2010) (link): 5′-TYGGTTTTGTYCCAACRATGG-3′ (forward degenerated primer) and 5′-CATAATCTACAGTGCCTTTCG-3′ (reverse primer). PCR was carried out in a Biometra Trio 48 thermocycler (Analytik Jena, Germany) in a final volume of 25 μl containing 1X NEB Q5 Reaction Buffer (New England Biolabs, Ltd, Ipswich, United States), 200 μM of each dNTP, 0.5 μM of each primer, 0.5 IU of NEB Q5 High Fidelity DNA Polymerase (New England Biolabs) and 1 μl DNA template. The temperature protocol included an initial denaturation at 98°C for 30 s followed by 30 cycles of 98°C for 10 s, 61°C for 30 s, 72°C for 30 s and a final extension at 72°C for 2 min. The INVISORB Spin DNA Extraction Kit (Invitek, Berlin, Germany) was used for DNA purification and products were sent to Eurofins Genomics Germany GmbH (Ebersberg, Germany) for sequencing using primer 5′-ATAATCTACAGTGCCTTTCG-3′ (Guinebretière et al., 2010 (link)). Sequence data analysis was done by uploading to an online algorithm1 for assignment to groups I to VII (Guinebretière et al., 2010 (link)).
Biometra trio 48 thermocycler
Manufactured by Analytik Jena
Sourced in Germany
The Biometra Trio 48 is a thermocycler designed for performing polymerase chain reaction (PCR) experiments. It features a 48-well block that can accommodate a variety of sample volumes. The Trio 48 is capable of precisely controlling temperature and ramping rates to ensure consistent and reliable PCR results.
Automatically generated - may contain errors
2 protocols using biometra trio 48 thermocycler
1
Bacillus panC gene classification
2
Chelex-Based DNA Extraction and Endosymbiont Detection
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DNA was extracted using the Chelex® method: A 5% Chelex® 100-resin (Bio-Rad, Germany) solution was made using UltraPure water (Invitrogen, Germany) and heated to c. 60 °C. 10 μl Chelex® solution and 1.5 μl Proteinase K (20 mg L -1 , Macherey-Nagel GmbH & Co. KG, Germany) were added to each sample. Aphids were homogenised using sterile pipette tips and mummies with a sterile micropestle. After homogenisation, 80 μl Chelex® solution was added, the sample was vortexed and incubated for 1.5 h at 56 °C. After incubation, samples were centrifuged at max speed for 5 minutes and the solution was transferred to a clean Eppendorf tube, diluted 1:2 with TE buffer (10 mM Tris-HCl pH 7.5; 1 mM EDTA pH 8.0), and stored at -20 °C. An extraction blank was included with each batch of DNA extractions.
Successful DNA extraction was confirmed using a PCR marker for the obligate endosymbiont B. aphidicola. The presence of facultative endosymbionts was determined using multiplex assays (Beekman et al., 2022) . Assays were conducted in a Biometra TRIO 48 Thermocycler (Analytik Jena, Germany). Primer and thermocycling details are described in Table S1. Successful amplification was detected by product separation on a 1% agarose gel stained with GelRed® (Biotium, Germany).
Successful DNA extraction was confirmed using a PCR marker for the obligate endosymbiont B. aphidicola. The presence of facultative endosymbionts was determined using multiplex assays (Beekman et al., 2022) . Assays were conducted in a Biometra TRIO 48 Thermocycler (Analytik Jena, Germany). Primer and thermocycling details are described in Table S1. Successful amplification was detected by product separation on a 1% agarose gel stained with GelRed® (Biotium, Germany).
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