DNA from bacterial isolates was extracted using the boiling method (Abrahamian et al., 2018 (link)) modified by the addition of a chloroform extraction. Single colony glycerol stocks were streaked and grown on nutrient broth yeast (NBY) medium for 48–72 h. Bacteria from a single colony was transferred into a 1.5 ml microcentrifuge tube and re-suspended in 400 ml of sterile water. Tubes were boiled at 100°C for 15 min in a dry-bath (Thermo Fisher Scientific, Waltham, MA, United States), and then immediately placed on ice for 5 min followed by centrifugation at 10,000 rpm for 5 min. An aliquot of 100 μL of isolated DNA was mixed with 300 μL of Tris EDTA buffer [10 mM Tris, 0.1 mM EDTA], and 400 μL of chloroform isoamyl alcohol (24:1) followed by centrifugation at 5,000 × g for 15 min. About 150 μL of the aqueous phase was transferred to a 96-well round bottom microtiter plate (Corning Inc., Kennebunk, ME, United States) containing 15 μL of sodium acetate (3M, at pH 5.3) and 330 μL of 70% ethanol followed by centrifugation at 5,000 × g for 10 min for DNA precipitation. Plates were air-dried and 100 μL of ddH20 was added. For DNA isolation of plant material we used methods described in Bernal et al. (2020) (link).
Dry bath
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dry Bath is a laboratory equipment used to incubate and maintain samples at a constant temperature. It provides a controlled and consistent heating environment for a variety of applications, such as cell culture, enzyme assays, and molecular biology procedures.
Automatically generated - may contain errors
Lab products found in correlation
96 well round bottom microtiter plate, by Corning (1 mentions)
Glycine, by Thermo Fisher Scientific (1 mentions)
Glycine, by Merck Group (1 mentions)
Ribo zero rrna removal kit for plant leaf, by Illumina (1 mentions)
Superase in, by Thermo Fisher Scientific (1 mentions)
Illustra microspin s 400 hr column, by GE Healthcare (1 mentions)
3 protocols using dry bath
1
Bacterial DNA Extraction with Chloroform
2
Glycine Crystal Preparation and Characterization
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Glycine (Sigma-Aldrich) was used without further purification. Glycine aqueous solution was prepared using ultrapure water (MilliQ). Glycine was dissolved at 80 °C using ultrasonication, filtered with a 0.2 µm Target2 Nylon syringe filter (Thermo Scientific), and then slowly cooled down (∼3 h) to room temperature in a dry bath (Thermo Scientific). The degree of SS was calculated using the solubility of Glycine in water at 20 °C (0.225 g mL−1) (56 ).
For SCNS, a sample was assembled by placing a silicone isolator sheet (0.25 mm thick; Grace Bio-Labs 664475) with a 10-mm hole on a piranha cleaned cover glass. The silicone sheet was cleaned in methanol before the use. Ten microliters of Glycine aqueous solution (SS = 1.2) was dropped in the hole, which resulted in a thin film of Glycine solution (100 µm thickness). Another cover glass was placed on top to prevent the evaporation of water. No spontaneous crystallization was observed for ∼1 h.
Conventional crystallization was performed to obtain α-, β-, and γ-Glycine, to measure Raman spectrum of each phase (SI Appendix, Fig. S1 ). α-Glycine was prepared by the slow evaporation of Glycine aqueous solution. β-Glycine was prepared by adding methanol to aqueous solution of Glycine. γ-Glycine was obtained by the slow evaporation of Glycine aqueous solution with potassium nitrate as additive (SI Appendix ).
For SCNS, a sample was assembled by placing a silicone isolator sheet (0.25 mm thick; Grace Bio-Labs 664475) with a 10-mm hole on a piranha cleaned cover glass. The silicone sheet was cleaned in methanol before the use. Ten microliters of Glycine aqueous solution (SS = 1.2) was dropped in the hole, which resulted in a thin film of Glycine solution (100 µm thickness). Another cover glass was placed on top to prevent the evaporation of water. No spontaneous crystallization was observed for ∼1 h.
Conventional crystallization was performed to obtain α-, β-, and γ-Glycine, to measure Raman spectrum of each phase (
3
Ribosome Profiling Protocol for RPF Isolation
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Prior to library construction, the RNA concentration of polysome extracts was first adjusted to 400 ng μL− 1. For isolation of RPFs, the 200 μL aliquots were subjected to nuclease (ARTseq/TruSeq Mammalian Ribo Profile Kit, ILLUMINA, USA) digestion (20 U per 40 μg of RNAs) at 25 °C for 1.4 h in a dry bath (THERMO, USA) with an agitation speed of 600 rpm. After terminating the digestion with 15 μL of SUPERase-in (AM2696, THERMO, USA), the resulting product was immediately placed on an Illustra MicroSpin S-400 HR column (GE HEALTHCARE, USA) that was equilibrated with 3 mL of PEB to collect the monosomes. RPFs were then extracted with the SDS method, and further purified by Zymo RNA clean and concentrator kits R1017 and R1015 (ZYMO RESEARCH, USA), respectively. The filtrate was subjected to rRNA removal with the Ribo-Zero rRNA Removal Kit for Plant Leaf (MRZPL1224, ILLUMINA, USA). Finally, RPFs around 28–30 nt were recovered by PAGE purification as described previously [73 (link)].
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