For the experiment depicted in Fig. 1a and Supplementary Fig. 9 , 24 surface-sterilized and stratified A. thaliana seeds were placed in two rows per 12 cm square plate containing ½ MS medium with 10 g/L Bacto-Agar (Duchefa Biochemie). Agar plates were sealed and incubated in a light cabinet under short day conditions (10 h light at 21 °C, 14 h dark at 19 °C) for 14 days. At day 14, plants were flushed with 15 mL 10 mM MgCl2 containing either live or heat-killed R401 WT cells at an OD600 of 0.0005 for 5 mins. Plants were transferred to new plates and grown for another 5 days for a total of 19 days. Shoot fresh weight was measured as a proxy to determine bacterial detrimental activity.
Bacto agar
Manufactured by Duchefa Biochemie
Sourced in Netherlands
Bacto Agar is a solidifying agent used in microbiological culture media. It is derived from red algae and provides a solid matrix for the growth of microbial cultures.
Automatically generated - may contain errors
4 protocols using bacto agar
1
Arabidopsis Seedling Bacterial Infection Assay
2
Arabidopsis T-DNA Mutant Genotyping
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Surface sterilized Arabidopsis thaliana seeds were sown directly onto half strength Murashige and Skoog (MS; Duchefa Biochemie, Netherlands), 0.8% Bacto Agar (BD). Stratification at 4 C in the dark was for 2 days, before transfer to Sanyo (UK) growth cabinets (19-22 C, 100 μmol m -2 s -2 ; 12 hr light/12 hr dark).
T-DNA lines used in this investigation are described in Table S1.
Genotyping was carried out according to the instructions at http:// signal.salk.edu/tdnaprimers.2.html using the primers listed in Table S2.
Left border PCR products were sequenced to determine the precise locations of the T-DNA inserts.
For srt1-4 heterozygous (hete) and srt1-4 hete x 2-1 mutants, we performed genotyping by PCR to identify heterozygous plants for each experiment. SRT1 artificial miRNAs were designed using the MicroRNA Designer tool of the WMD3 Web site (Ossowski, Schwab, & Weigel, 2008) and were introduced by transformation as described below.
T-DNA lines used in this investigation are described in Table S1.
Genotyping was carried out according to the instructions at http:// signal.salk.edu/tdnaprimers.2.html using the primers listed in Table S2.
Left border PCR products were sequenced to determine the precise locations of the T-DNA inserts.
For srt1-4 heterozygous (hete) and srt1-4 hete x 2-1 mutants, we performed genotyping by PCR to identify heterozygous plants for each experiment. SRT1 artificial miRNAs were designed using the MicroRNA Designer tool of the WMD3 Web site (Ossowski, Schwab, & Weigel, 2008) and were introduced by transformation as described below.
3
Storing and Culturing Bacterial and Fungal Samples
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Bacteria were streaked from glycerol stocks (25% glycerol) on TSA plates (15 g/L Tryptic Soy Broth, Sigma Aldrich; with 10 g/l Bacto Agar, Duchefa Biochemie) and grown at 25 °C. Single colonies were inoculated in liquid 50% TSB (15 g/L Tryptic Soy Broth, Sigma Aldrich) and grown until dense at 25 °C with 180 rpm agitation. Dense cultures were then stored at 4 °C and diluted 1 to 10 in TSB the day before the experiment and cultured at 25 °C with 180 rpm agitation overnight to ensure sufficient cell densities for slow- and rapidly-growing bacteria. Glycerol stocks were stored at -80 °C and kept on dry ice when transported. Individual pieces of fungal mycelium were transferred to potato dextrose agar (PDA; Sigma-Aldrich) Petri dishes from glycerol stocks (approx. 30 pieces of fungal mycelium in 25% sterile glycerol, stored at -80 °C). Fungi were grown at 25 °C in the dark for 14 days.
4
Antimicrobial Assessment of Lactic Acid Bacteria
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To determine the antimicrobial activity of LAB cultured in MRS or juice, spot‐on‐the‐lawn assays (Lee & Chang, 2016 ) or paper disk assays (Yang & Chang, 2010 ) were used. Briefly, LAB strain was cultivated in MRS broth at 30℃ for 24 hr or in juice at 15℃ for 0–5 days. The culture was then centrifuged (10,100 g, 15 min, 4℃) and the supernatant sterilized by passing through a 0.45 μm membrane filter (Sartorius) and used as culture filtrate or juice filtrate.
The prepared culture filtrate or juice filtrate was used for the antimicrobial activity assay. Plates were prepared by adding fungi (6.0 log spore per 20 ml MEA or PDA) to 1.5% bacto agar (Duchefa) for antifungal assays or by spreading the bacteria (6.0 log CFU/mL) onto MRS, LB, TSB, or NB + 2% NaCl agar for antibacterial assays (Lee & Chang,2016 ; Yang & Chang, 2010 ). For the paper disk assay, paper disks (diameter 8 mm; Advantec, Tokyo, Japan) on MRS plates were spotted with 100 µl of sample. The plates were then incubated at 30℃ for 24 hr and examined for inhibition zones. For the spot‐on‐the‐lawn assay, 10 µl of sample was spotted onto the sensitive mold and bacterial plates. Antimicrobial activity, which was defined as the reciprocal of the highest dilution at which microbial growth was inhibited, was expressed as arbitrary units (AU) per milliliter.
The prepared culture filtrate or juice filtrate was used for the antimicrobial activity assay. Plates were prepared by adding fungi (6.0 log spore per 20 ml MEA or PDA) to 1.5% bacto agar (Duchefa) for antifungal assays or by spreading the bacteria (6.0 log CFU/mL) onto MRS, LB, TSB, or NB + 2% NaCl agar for antibacterial assays (Lee & Chang,
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