Plates from Ahringer’s RNAi library containing the clones of interest (purchased from the Medical Research Council (MRC) GeneService) were replicated to Luria-Bertani (LB) agar supplemented with ampicillin (100 μg/ml, Sigma-Aldrich) and tetracycline (15 μg/ml, Sigma-Aldrich) using a pin replicator (Boekel) and grown overnight at 37 °C. The day after, the selected clones were inoculated in 1.3 ml of LB supplemented with ampicillin (100 μg/ml, Sigma-Aldrich), tetracycline (15 μg/ml, Sigma-Aldrich) and 8% glycerol in deep well plates (VWR) and incubated overnight at 37 °C with shaking (180 rpm, New Brunswick™ Innova® 44/44R incubator shaker). The last column of the plates was left free for convenient control. The next day, 120 μl of the culture was transferred to microtitre plates using a Precision XS Microplate Sample Processor (Biotek, Winooski, VT, USA) and frozen at −80 °C.
Deep well plates
Manufactured by Avantor
Deep-well plates are a type of laboratory equipment used for sample preparation, storage, and processing. These plates feature multiple individual wells that can hold larger volumes compared to standard microplates. The depth of the wells allows for increased sample capacity, making them suitable for applications that require larger sample sizes or reaction volumes.
Automatically generated - may contain errors
Lab products found in correlation
Tetracycline, by Merck Group (2 mentions)
Ampicillin, by Merck Group (2 mentions)
Precision xs microplate sample processor, by Agilent Technologies (1 mentions)
Ampicillin, by Avantor (1 mentions)
Tetracycline, by Avantor (1 mentions)
Innova 44 44r, by Eppendorf (1 mentions)
96 well plate, by Avantor (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Carbenicillin, by Merck Group (1 mentions)
5 protocols using deep well plates
1
Preparation and Storage of RNAi Library Clones
2
Scalable RNAi Library Preparation
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OrthoList RNAi plates were replicated in LB agar, and bacteria were grown overnight at 37 °C. The advantage of growing the bacteria in solid media is the ease of visualising the clones where the bacteria did not grow. If needed, the samples can be kept at 4 °C for 2 days maximum. Next day, we inoculated the RNAi library in 2.2-ml 96-well plates (VWR). Using the pin replicator, we inoculated the bacteria from the LB agar into 1.2 ml of LB supplemented with ampicillin (100 μg/ml, Sigma-Aldrich) and tetracycline (15 μg/ml, Sigma-Aldrich). Positive and negative controls were added in the last column of the plate. We cultured the bacteria overnight at 37 °C with shaking (180 rpm, New Brunswick™ Innova® 44/44R). In order to have fresh cultures, on the day of sorting, we inoculated 100 μl of the O/N cultures in 900 μl of LB supplemented with ampicillin and tetracycline in deep well plates (VWR) and incubated for 3 h at 37 °C with shaking. We added isopropylthio-β-galactoside (IPTG, 1 mM, Sigma-Aldrich) to the wells to induce the expression of the plasmid for 2 h at 37 °C with shaking. Then, we harvested the cultures by centrifugation (10 min, 3200 g, 4 °C, Eppendorf 5810R) and re-suspended the pellets in 250 μl of S-medium supplemented with carbenicillin (25 μg/ml, Sigma-Aldrich), IPTG (1 mM, Sigma-Aldrich) and cholesterol (5 μg/ml, Sigma-Aldrich).
3
Metabolomic Analysis of E. coli Interactions
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Overnight cultures of E. coli BW25113 harbouring either BAC-pks or the empty BAC were back-diluted 1:100 and co-cultured in M9-CAS at a 1:1 ratio with phage-free E. coli BW25113 or lambda-infected BW25113. Cultures (1 ml) were dispensed into deep-well plates (VWR) and incubated with shaking at 37 °C. After 24 h, 10 µl deuterated (d27) N-myristoyl-d -asparagine (10 µM in DMSO stock solution) was added to each sample. Samples were flash-frozen in liquid nitrogen, lyophilized for 48 h, then reconstituted in methanol (1 ml) and vortexed for 1 min. Three hundred microlitres of the mixture was filtered through a 0.22 µm filter (Pall) before mass spectrometry analysis.
4
High-throughput Yeast Stress Response Assay
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YKO collection strains were pinned onto YEPD agar plates using a Singer ROTOR robot and grown for 2 d at 30°. The yeast were then pinned from the agar plates into 96-well plates containing 100 μl of YEPD per well and grown for 18–22 hr at 30°. The overnight cultures were then used to inoculate 2.2 ml deep-well plates (VWR), containing 550 μl of YEPD, and one sterile 3.2-mm stainless steel mixing bead per well, to an OD600 of 0.05, and loaded into the Liconic Incubator (shaking at 1200 rpm and 30°). Once the median OD600 of a plate reached 0.60 (no wells reached an OD600 of > 0.8), 150 μl of each culture was transferred to a 2.2 ml 96-well plate containing 850 μl of RNAse Inactivation Buffer per well (RI Buffer; 4 M ammonium sulfate, 100 mM MES buffer, and 20 mM EDTA, pH 4.6), and mixed thoroughly by pipetting; 100 μl of 1.875 M KCl, or 1 μg/ml rapamycin in 30° YEPD was then added to each remaining culture (yielding final concentration of 0.375 M KCl or 200 ng/ml rapamycin), and the plate was returned to the incubator for 19 min (shaking at 1200 rpm and 30°). The plate was then moved back to the deck of the robot, and 150 μl of culture removed from each well and added to RI Buffer as described above. The plates containing RI Buffer and yeast were then stored at –20°.
5
Catechin Conversion Protocol
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Cells were grown overnight at 37°C in 1 mL LB medium supplemented with 80 μg/mL ampicillin and 25 μg/mL chloramphenicol in 48-deep-well plates (VWR). Overnight cultures (20 μL) were sub-cultured into 1 mL of fresh LB medium containing the necessary antibiotics in 48-deep-well plates. Cells were grown at 37°C with shaking at 220 rpm, induced with 0.5 mM IPTG when OD600 (the optical density at 600 nm) reached 0.6, and further grown at 30°C with shaking for 3 h. Cells were then collected by centrifugation at 4,000 rpm and 4°C and resuspended in 1 mL of M9 medium (pH 5.0) with 500 mg/L (+)-catechin. The conversion process was then carried out at 30°C with shaking for 19 h. At the end of the fermentation, 200 μL of the culture supernatants were mixed with an equal volume of acidified methanol and centrifuged at 12,000 rpm for 10 min. The supernatants were used to quantify residual catechin and extracellular C3G.
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