| Property | Value |
|---|---|
| BioDare ID | 2842 |
| Author | Anna Flis |
| Institution | MPI of Molecular Plant Physiology |
| License | CC_BY |
WT and elf3-4 mutants, in biological duplicates, diurnal light, harvest every 2 hours Numbers are in transcript copy per cell, obtained assuming 1 g FW contains 25000000 cells.
TiMet focuses on an important and general biological problem – how are plant metabolism and growth regulated and orchestrated in a daily-changing environment?
The elf3 mutant was harvested at 13 day not at 21 as the rest due to its early flowering phenotype. This experiment is partenr to "TiMet WP1.1 qRT-PCR LD to LL and LD"
Plant material was grinded using Ball-Mill (Retch, Germany). Around 50 mg of material from each sample was aliquoted into 2 µL Eppendorf tubes. RNA was extracted using RNeasy Plant Mini Kit (QIAGEN) by following the manufacturer’s instructions. Briefly, the RLC buffer (500µL) was added to the frozen plant powder and the mixture was homogenised. RNA was eluted from the RNeasy spin column twice, first with 50 µL and then with 30 µL of RNase-free water. The concentration of RNA was determined using the Nano-Drop ND-1000 UV-Vis spectrophotometer (Nano-Drop Technologies).
RT-PCR (PerkinElmer Evolution P3+Applied Biosystems PCR)
In the first step DNA that may be present in RNA extracts was removed from samples using TURBO DNA-freeTM kit (Applied Biosystems) by following the supplied protocol. The concentration of RNA was determined using Nano-Drop spectrophotometer and 1µg of DNase treated RNA was used for cDNA synthesis. The reaction of reverse transcription was done using the SuperScript III First-Standard Synthesis System Kit (Invitrogen).
On each 384-well plate with an ABI PRISM 7900 HT sequence detection system (Applied Biosystems Deutschland, Darmstadt, Germany) using PerkinElmer Evolution P3 Precision Pipetting Platform (PerkinElmer Life Science, Rodgau-Jügesheim, Germany) 22 pairs of primers were dispensed (200nM of each primer). PCR mix was prepared by adding 0.5µL of cDNA and 2.5µL of Power SYBR Green PCR Master Mix (Applied Biosystems, Deutschland) to already prepared primers. The qRT-PCR reactions were carried out following the recommended thermal profile: 2 min at 50˚C, 10 min at 95˚C, followed by 40 cycles of 95˚C for 15 s and 60 ˚C for 1 min. The specificity of the amplifications was tested by heating from 60˚C to 95˚C with a speed of 1.9 ˚C min-1, resulting in melting curves. Data analysis was performed using SDS 2.4 software (Applied Biosystems Deutschland).
Done by Robot: PerkinElmer Evolution P3 Precision Pipetting Platform with Applied Biosystems PCR, PRIMERS: first forward then reverse LHY TGCCTCAAAGCTTTTCGCCTCCT GTCTGCAGCACAAGAATCCTGGCT CCA1 TCCAATGCACGCCGCAGTAGAA AGGCAATTCGACCCTCGTCAGACA PRR9 AGCTAGCAGAACAACGTCCTCGAGT CGTCTGAATTCACGGTTCGCACGA PRR7 AGAGGTGCTTCCGAAAGAAGGTACGA ACGCACAAATTGGCCTCGCACT PRR5 TGCAATGGCTCCTGCTTCACTCTC ACTGTACTCATGCGGGCTAACGGA TOC1 TGATGGATCGGGTTTCTCTGCACCA TGAGGCATCATGGCTGCTGATTGC GI TGCGGGCAACTGATGGAATGCT TGCTCTTGCCGTGGCTTCAAGT LUX CAGCGGTAATGTTGGAGTGCCGAT TGGCATCTGCATCATCTGTTGCGT ELF3 ACAACAAGAGATGGGGGAGGAGTGAC ACTCGCGAGCTTTGCGTTGTGA ELF4 AGTTTCTCGTCGGGCTTTCACGGT TAAGCTCTAGTTCCGGCAGCACCA
Growth on Soil for 12.0 days (LD 20/18C). ELF3 was gorwth for shorter than other mutants to avoid bolting
| Type | Duration (days) | Cycle (h) | Start | Duration | Spectrum | Source | Intensity |
|---|---|---|---|---|---|---|---|
| diurnal light | 12 | 24 | 0:00 | 12:00 | white | tube | 160 |
| Type | Duration (days) | Cycle (h) | Base (°C) | Warm (°C) | Warm start | Warm duration |
|---|---|---|---|---|---|---|
| diurnal temp | 12 | 24 | 18 | 20 | 0:00 | 12:00 |
Growth on Soil for 2.0 days (LD 20/18C). Continuation of the growth conditions, harvested only 1 day
| Type | Duration (days) | Cycle (h) | Start | Duration | Spectrum | Source | Intensity |
|---|---|---|---|---|---|---|---|
| diurnal light | 2 | 24 | 0:00 | 12:00 | white | tube | 160 |
| Type | Duration (days) | Cycle (h) | Base (°C) | Warm (°C) | Warm start | Warm duration |
|---|---|---|---|---|---|---|
| diurnal temp | 2 | 24 | 18 | 20 | 0:00 | 12:00 |