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Arabidopsis - Science method

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I performed micrografting on mutant for ABA transport and most of them didn't succed. I'm considering that the mutation might have hindered the vascular regeneration. Do you know how ABA could affect vascular regeneration?
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Nicolò Maria Villa ABA regulates root growth, especially defining the directions in which the root system spreads. Because the development of vascular elements is important for root growth, ABA necessarily also affects the development of vasculature. There might be a specific interaction between the particular ABA transporter in your research and some element of the vascular development cascade - that interaction may or may not be already researched, so you would need to take a thorough look at the literature. Maybe you might start from the following:
And, possibly:
and see where that will lead you.
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I am isolating plasmid DNA using the alkaline lysis method followed by an additional purification step of Ammonium acetate precipitation.
I am isolating protoplast using Sheen protocol.
I am using 40% PEG 4000 and 20 ug of plasmid DNA. I have tried different transformation periods ranging from 5-30 mins but there is no transformation at all.
My protoplasts stay healthy up to 72 hrs even after DNA PEG treatment but none of them are getting transformed.
Any help will be most appreciated.
Thanks
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@hendry susila,
Thank you for the answer.
The plasmid concentration is around 4ug/ul.
I am transferring around 20 ug in total.
All the clones are in pUC19 so size is not an issue.
I tried empty gfp vector too, same problem there as well
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Hello, I'm running plant chIPs and I've heard it is possible to grind the tissue and add it directly to the first extraction buffer mixed with formaldehyde skipping the vacuum infiltration step, but I don't see any written protocol on this. If I do this, should the extraction buffer be at Room Temp or cold? Does this change anything else I need to do in the overall protocol?
What general temperature should I work at with chip buffers from lysis to washing? Is room temp fine or should it be chilled?
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Dear Ad Ad ,
In order to respond properly, I'd need to know which plant you're working on, what's your purpose, the condition of your samples and what kit you're using.
Generally, colder is better simply because it slows down the metabolism, including the enzymes that you release after grinding and stabilises the nucleic acids. I generally work in a cold room and keep all the samples and reagents under ice. Also, always keep in mind that room temperature is not a standard at all. It is generally used when the temperature doesn't matter within an interval 10-40°C, which in biology is rare.
Anyway, I doubt that skipping the vacuum infiltration is a good idea because the plant cell doesn't allow for an easy diffusion due to rigid cell walls, high levels of cellulose and lignin, large vacuoles etc. Moreover, you need to consider the possible presence of air bubbles within tissues and among tissues, in particular inflorensces. Vacuum infiltration is applied to allow the formaldehyde to penetrate into plant cells and organelles (incl. nuclei). Finally, if you had the formaldehyde after grinding, you'll "snapshot" a different profile because you already damaged mechanically and, even if you add it before grinding, the lysis buffer is not entirely and immediately effective.
Why do you want to skip the vacuum infiltration?
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Hi, I'm finishing my thesis and I need to store freshly harvested Arabidopsis seeds.
Is it better to sterilise them, let them dry and store them or let them dry, store them and remind the future user of sterilising them before use?
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It very much depends on the sterilisation method you are using. If it is sodium hypochlorite, then you can't really store sterilised seeds since it is hard to properly dry them and they would be unusable fast if left moist. I personally use an ethanol method that allows the seeds to completely dry after sterilisation and I usually keep them for a long time. Their germination rate doesn't seem to be affected in any way. However, I only sterilise a small batch and not all my seeds. I store them in paper bags or glass vials and make sure they are dry.
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Can you please recommend some publication where is argued the use of arabidopsis as model for crops?
For my thesis I compared the results between Arabidopsis and a crop to assess how representative is Arabidopsis as a model. I need some more papers for the discussion of the subject.
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Try to check that with different titles on Google. Regards.
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In our lab we usually dry arabidopsis seeds(Col-0) in 37 degree incubator after haversting. But I found several protocols in which drying seeds in room-temperature. So im worried about whether 37 degree is too high for arabidopsis seeds?
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As Yuan-Yeu Yau wrote, it leads to heat shock and that definitely alters the expression profile. Thus, if you do it, you must do it for all the seeds that you're going to use in the experiment, unless the drying temperature is part of the treatment in your experimental design, and explicitly mention it in the material & methods.
Although the seeds are most likely viable after drying at 37°C, it's not the method that ensures the highest viability and germination rate. You must also consider the ecotypes and eventual mutations. For example, seeds of the abscisic acid-insensitive (ABI) mutants fail to degrade chlorophyll during maturation and show no dormancy, leading to low desiccation tolerance and poor longevity.
According to the Arabidopsis protocols, the recommended method is to air-dry the seeds at room temperature and about 20% RH for 1–3 weeks. I stop watering my plants for the 1-2 weeks after they developed the siliques and afterward I harvest them. Then you pack the seeds in cryovials and store them at 4°C at max 30% RH for up to 2 years or at -20°C and max 20% RH for longer. You also must make sure that they dry well after sterilisation. Whether is better to sterilise them immediately or only before immediate use it's debated. Seed moisture content should be around 5% and there are several methods of measuring it.
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I would like to use STRING for functional analysis of proteome data, as it appears to be quite powerful. Unfortunately I'm working with an organism that isn't in STRING's database, nor is there anything particularly closely related on the species list. STRING requires all proteins to be from a single species on their list. Is there a sound method to use my data in STRING, perhaps using gene orthologs from another species? For example, could I BLAST my differentially expressed protein sequences against the genome of a single model organism (e.g. Arabidopsis) and use the top hits as the gene IDs? I've tried using simple gene names as the input, but typically only about half are present in any single model organism I've tried. I'm aware that there are hazards in using orthologs, but I'm mostly interested in higher-order patterns and GO term enrichment.
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you may try to find the homologous genes in another organism, or use the domain-domain approach ..based on Pfam
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I need to enrich my MS/2 medium with 0.1 and 0.5 uM ABA to assess it's effect on the roots of some Arabidopsis mutants.
I tried by simply adding an aqueous solution on top of the medium, but I'm afraid is causing an increase in mobility of the seeds and contamination. I don't think I can add it before because the temperature of molten MS might damage ABA while the ABA solution would cool down the MS non-homogeneously. Any other idea?
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Do you pour the ABA in powder directly in the ABA or do you add it as a solution?
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Hello everyone,
I have planned to start the experiment for DAB staining in Arabidopsis leaves. Followed by staining I need to quantify the intensity of developed color through NCBI image J software.
I would be thankful to anyone who can send me the appropriate answer with the instructional command in software for intensity measurement and analysis.
What unit I should use to plot the bar graph for the measurement?
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Hi Amber Gupta. Measuring the intensity of immunostained (e.g. DAB) slides is not a useful parameter due to the stoichiometric nature of such stains. It might be better to quantify the DAB-positive area fraction or count the number of DAB-stained cells. In any case, you will have to segment this area using color deconvolution.
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When we used 35S(pBinGlyRed) to construct overexpressing Arabidopsis, we found several plants emitting red light in T1 generation by green light irradiation. This has never been found in our previous research. We want to find out why. Is this phenomenon common?
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Ah, you are observing variability in the T1 plants (some are completely bright red, others are less intense). You may have multiple insertions and/or position effects influencing the expression levels.
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Hello Everyone,
What should I do to get rid of the RuBisCo band from Arabidopsis tissue sample?
1. My target protein was extracted from Arabidopsis tissue. I loaded 50 ug of protein. After transferring the gel to PVDF membrane, I did Ponceau S staining to confirm the transfer. There was a RuBisCo band along with another non-specific band after staining.
2. I kept blocking at RT for 2 hrs.
3. My protein was tagged by GFP and I used GFP monoclonal primary antibody (1: 5000 dilution), 4 degree overnight, and appropriate compatible secondary antibody-HRP (1:10000 dilution) for 2 hrs at RT.
I didn't get a band for my target protein, only getting the RuBisCO band after developing the blot.
Does anyone face this kind of problem or suggest me how to get rid of this?
Thank you in advance!
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I had the same problem and found the solution.
There is a very good antibody from Agrisera that does not give rubisco unspecific band. It´s the anti C-YFP antibody from Agrisera.
It recognizes only the C-terminal half of YFP, CFP or GFP and it works perfectly fine to detect the whole protein. I struggled a lot until I found this antibody and now doing WB is a pleasure (well... still tedious and all but gives nice bands :P)
check the blot to see how nice it works
As secondary I´m using anti-rabbit-HRP from santa cruz (a cheap one)
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In plants, phyohormones induced responses generally see the binding of transcription factors to the special motifs present in the promoter of a downstream gene. After a few preliminary results, I was checking if the well known DNA binding element is present in my gene's promoter or not. Although I found a few variant of the original motif sequence in the promoter region, the exact motif which I was looking for I found is present in some +100 base pairs down to the start codon. In my understanding as far as I know, the DNA binding elements (motifs) are generally found in the upstream of the protein encoding region (promoter usually). But does anyone knows about cases where transcription factor binding sites, the DNA motifs are also present inside the gene itself?
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There are many, many examples of TF binding sites in the gene body. After the first whole genome ChIP experiments (ChIP-chip and ChIP-Seq) , it became obvious.
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Currently I keep them on perlite with irrigation using Summerfield solution, however the plants I get are 0.5cm in diameter and very weak even the wild ColO phenotype. These plants produce few seeds, not optimal. Can anybody help me please?
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Any reason why you can't grow your plants in soil with fertilizer, vermiculite and perlite? The plants tend to perform better when grown in soil than in sterile culture.
If they do need sterile culture, check the lighting conditions. 16 hours light 8 hours dark is pretty standard. Etiolated growth indicates poor light intensity/quality, purple color in the leaves indicates too high of intensity (or high temperature)
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Has anyone else dealt with static electricity on Arabidopsis plates? The static causes the seedlings to root upwards. Passing your finger along the top of the plate causes the upward root to follow your finger and move. Sometimes the entire seedling will lift off of the media and stick to the lid of the plate. Since the seedling is not rooting properly, it makes drug selection difficult.
Media: 1/2MS + 0.6% agar + BASTA
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The best way to deal with static charge is increasing humidity. So if you're not afraid of contamination, you should put a humidifier in the culture room where you grow the Arabidopsis.
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I've received seeds of Arabidopsis col-0 and ws mutants for nitrate uptake, and they don't germinate.
Seed conditions
They're as old as 13 years and were kept in airtight plastic bag at room temperature.
Sterilisation
I sterilised them by shaking them for 5 minutes in a water solution 50% EtOH (v/v), 1.2% NaOCl (v/v, bleach), then rinsed them 3 times with EtOH 99%, and 3 times with autoclaved milliQ dH2O and left them dry on autoclaved filter paper under sterile hood. The floating seeds are removed during rinsing.
Sowing
I sowed them on MS/2 in Petri dishes and sterilised soil.
The ones on MS/2 were stratified at 4°C for 4 days, a second batch for 1 week.
Most genotypews have not germinated on either MS/2 nor soil.
I tried to add GA4+7 10 uM to the MS/2, but didn't induce germination.
Could you suggest any idea to try induce germination and flowering?
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Firstly, test seeds viability via tetrazalium.
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In 2001, Lin et al. pointed out that there are at least 17 heat shock proteins (HSPs) in the Arabidopsis genome, among which the accession number of HSP70-17 is BAC Z97341. I found the corresponding protein sequence in NCBI, and its accession number is GenBank: AAL38281. 1. But this gene is annotated as a heavy metal transporter, not the heat shock protein I am looking for. Please tell me, how can I find the ID number and protein sequence of HSP70-17? Article information is shown below.
Lin B L , Wang J S , Liu H C , et al. Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana[J]. Cell Stress & Chaperones, 2001, 6(3):201-208.
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The annotations are a "best guess". If the protein sequence & DNA are a match, then that's your gene. Lots of what we classically call "heat shock proteins" might be better named "stress induced proteins" since many factors light heat, cold, salt, and toxins will also increase their expression.
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Can you recommend some publication where is argued the use of arabidopsis as model for crops?
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Arabidopsis thaliana has become universally recognized as a model plant for such studies. Although it is a non-commercial member of the mustard family, it is favored among basic scientists because it develops, reproduces, and responds to stress and disease in much the same way as many crop plants. What's more, Arabidopsis is easy and inexpensive to grow, and produces many seeds; this allows extensive genetic experiments, often involving tens of thousands of plants. Also, Arabidopsis has a comparatively small genome, thereby simplifying and facilitating genetic analysis. Compared to other plants, it lacks the repeated, less-informative DNA sequences that complicate genome analysis.
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How to desing gRNA for CRISPER/Cas9 KO to target two domains of proteins?
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Targeting the promoter is risky as you will still have the actual gene present. I would go for target sites in the gene itself, try for a very large deletion. Some key considerations
make sure your target sites are unique
if possible get target sites that are not in the same reading frame (or you have a higher chance of a in-frame deletion)
use the gene sequence for your ecotype of Arabidopsis for your target site selection and gRNA design
Hope this helps!
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I am planning to do a lot of ChIPs following 'Chromatin techniques for plant cells Bowler 2004' and thus I am planning on stockpiling a lot of stock solutions and premade ChIP buffers. I'm having a very difficult time finding any consistent info on storage conditions for even individual solutions and whether they should be sterile and whether to do it by autoclave or filter. Multiple sources give conflicting information so I've tried to compile what I felt was the consensus below.
I was wondering if I could get any help confirming that the below information is correct and fill in the missing info?
A: typical storage condition/time in storage
B: storable at -20C?/time stable at -20C
C: storable at -80C? /time stable at -80C
D: light sensitive?
E: should be autoclaved?
F: should be filter sterilized?
G: special considerations
Stock Solutions
Formaldehyde 37% :
A: RT/2 yr B: Yes/indefinite C: Yes/indefinite D: NO E: NO (obviously) F: NO G: hazardous
Glycine 2M :
A: 4C/2 yr B: Yes/indefinite C: Yes/indefinite D: NO E: YES (autoclave or filter sterilize) F: YES
SUCROSE 2M :
A: RT/6m B: Yes/indefinite C: Yes/indefinite D: NO E: YES (autoclave as long as solution does not turn brown or filter sterilize) F: YES
Tris-Hcl pH 8 1M :
A: RT/indefinite B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: NO
B-ME 14.3M:
A: RT/? B: Yes/indefinite C: Yes/indefinite D: ? E: NO F: NO G: hazardous
PMSF 0.2M :
A: -20C/? B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: NO G: hazardous
MgCl2 1M:
A: RT/indefinite B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: NO
Triton X 20%:
A: RT/indefinite B: ? C: ? D: NO E: NO F: NO
EDTA 0.5M:
A: RT/indefinite B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: NO
20% SDS
A: RT/indefinite B: No/precipitates C: No/precipitates D: NO E: NO F: NO
NaCl 5M:
A: RT/indefinite B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: NO
NaHCO3 :
A: RT/indefinite B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: NO
NP-40:
A: RT/indefinite B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: NO
LiCl 4M: A: RT/indefinite B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: NO ChIP Solutions Extraction Buffer 1 0.4 m Sucrose 10 mm Tris–HCl, pH 8.0 5 mM β-ME 0.1 
A: 4C/6m? B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: YES
with sodium butyrate 5mM
A: 4C/6m? B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: YES
Extraction Buffer 2 0.25 M Sucrose 10 mm Tris–HCl, pH 8.0 10 mM MgCl2 1% Triton X-100 5 mM β-ME
A: 4C/6m? B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: YES
with sodium butyrate 5mM
A: 4C/6m? B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: YES
Extraction Buffer 3 1.7 M Sucrose 10 mm Tris–HCl, pH 8.0 0.15% Triton X-100 2 mM MgCl2 5 mM BME
A: 4C/6m? B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: YES
with sodium butyrate 5mM
A: 4C/6m? B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: YES
Nuclei Lysis Buffer 50 mM Tris–HCl, pH 8.0 10 mM EDTA 1% SDS
A: RT/1yr? B: Yes/indefinite C: Yes/indefinite D: NO E: YES F: YES
with sodium butyrate 5mm
A: RT/1yr? B: Yes/indefinite C: Yes/indefinite D: NO E: YES F: YES
ChIP dilution buffer 1.1% Triton X-100 1.2 mM EDTA 16.7 mM Tris–HCl, pH 8.0 167 mM NaCl
A: RT/1yr? B: Yes/indefinite C: Yes/indefinite D: NO E: YES F: YES
with sodium butyrate 5mm
A: RT/1yr? B: Yes/indefinite C: Yes/indefinite D: NO E: YES F: YES Elution buffer 1% SDS 0.1 m NaHCO3
A: RT/4hr B: Yes/indefinite C: Yes/indefinite D: NO E: NO F: ? Low salt wash buffer 150 mM NaCl 0.1% SDS 1% Triton X-100 2 mm EDTA 20 mm Tris–HCl, pH 8.0
A: RT/indefinite B: Yes/indefinite C: Yes/indefinite D: NO E: ? F: ? High salt wash buffer 500 mM NaCl 0.1% SDS 1% Triton X-100 2 mm EDTA 20 mm Tris–HCl, pH 8.0
A: RT/indefinite B: Yes/indefinite C: Yes/indefinite D: NO E: ? F: ?
LiCl wash buffer 0.25 M LiCl 1% NP-40 1% sodium deoxycholate 1 mM EDTA 10 mm Tris–HCl, pH 8.0
A: RT/indefinite B: Yes/indefinite C: Yes/indefinite D: NO E: ? F: ?
TE buffer 10 mm Tris–HCl, pH 8.0 1 mM EDTA
A: RT/indefinite B: Yes/indefinite C: Yes/indefinite D: NO E: ? F: ?
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Dear AdAd,
that's kind of an unwieldy question. A lot of it is self-explanatory.
Let me see:
Formaldehyde 37% :
best to prepare fresh from paraformaldehyde
Glycine 2M :
filter sterilize, RT
SUCROSE 2M :
autoclave, RT
Tris-Hcl pH 8 1M :
autoclave, RT
B-ME 14.3M:
as given by supplier (4C), no need to sterilize
PMSF 0.2M :
make in isoprop, store at -20C, once in use at RT, decays in aqueous solutions within minutes!
MgCl2 1M:
autoclave, RT
Triton X 20%:
no need to do anything, RT
EDTA 0.5M:
autoclave, RT
20% SDS
come on, 20% SDS???? no need to do anything, nothing will grow
NaCl 5M:
autoclave, RT (try at 4C and you will see how you get very nice crystals....)
NaHCO3 :
prepare fresh, solutions decay by exchange with atmospheric CO2
NP-40:
no need to do anything, RT
LiCl 4M: autoclave, RT
For your working solutions it is best to prepare them fresh for each experiment with your stocks. That's easy and keeps contaminations away.
Good luck with your experiments.
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Hello everyone!
My protein which has a GFP tag is well expressed in nuclei. To visualize cell boundaries in different layers of the root sample, I am planning to counter stain my samples with propidium iodide and DAPI.
I will be taking images using 3i spinning disc confocal microscope.
I want to know if anyone has a working protocol for such settings.
Earlier, I tried Hoechst 33342 to stain nuclei but it appears that even incubating for 30 minutes, the stain was not able go inside the cells as the cell boundaries were clearly visible during microscopy. I tried DAPI as well, but the background was very high even after 3-4 washes and the intensity of nuclei was not great.
I will appreciate if you could share with me your experiences and suggestions regarding my question.
Thanks!
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Hi, are you planning to use the propidium iodide for both cell boundaries and nuclei?
If your GFP tag is well expressed in the nuclei, you do not need to stain it again. But if the fluorescence signal is not as bright as you want it to be, open the pinhole for more light to reach your sample.
I have used propidium to stain the cell boundary of Arabidopsis roots in the past, and it worked brilliantly. I stained the root for just 5 minutes using the recommended concentration on the bottle.
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I have in my RNA-seq quantification data from Arabidopsis obtained by mRNA-seq polyA enrichment library transcripts encoded by chloroplast and mitochondrial genes in significant DEG. How is it possible, if chloroplast and mitochondrial transcripts do not have poly-A tail? Are these data reliable or contaminants which should be ignored?
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I checked it and I probably found the response: Moreover, polyadenylation is often
a degradation signal in organelles, meaning that researchers
using poly(A)-selected RNA-Seq for measuring differential
expression in organelle systems may, in some instances,
be measuring the opposite: differential degradation (Smith and Lima, 2016).
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Hi,
I am going to transform overexpression gene construct into Arabidopsis in future. My question is which background genotype of Arabidopsis I should use for this transformation and why? thanks in advance
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"The most commonly used wild type is Columbia-0 (Col-0); Landsberg erecta (Ler) and Wassilewskija (Ws) are also commonly studied."
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I am working in drought and salt stress with Arabidopsis and Tomato. I need to give these stress conditions to minimum 2 week old seedling.
Could I give these stress treatment in soil by giving equal volume of PEG or sorbitol solution (drought) and NaCl solution (salt) and water (Control)?
I am more doubtful in case of drought treatment instead of giving dehydration treatment could I follow sorbitol treatment in soil?
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Please have a look to the following article for your reference.
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I need Arabidopsis cells in suspension for transformate them with agrobacterium.
Now, my line of Arabidopsis is growing in MS Agar media in plates forming callus.
I'm looking for a method to transfer this callus to a liquid media.
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Exactly, we don't have the seeds. At this moment, we aren't gonna grow plants. Probably later.
Thank you!
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I will sequence the micro RNA isolated from Arabidopsis and want to detect the presence (if any) of small RNA which are nor originated from Arabidopsis genome. Please provide me your valuable suggestions for this study. Thanks
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If your are using STAR to align your miRNA you can use the --outSAMunmapped or --outReadsunmapped options to get unmapped reads
Here's the manual for further information:
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I want to confirm protein localisation in Arabidopsis using fluorescent protein-tag. What are proper markers for plasma membrane, apoplast and cell wall?
Would anybody share such lines or plasmids with such markers?
Thank you.
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I agree with the GFP-PIP2 suggestion above. Cell walls can be stained with various dyes (here is one option)
A protocol for combining fluorescent proteins with histological stains for diverse cell wall components - Ursache - 2018 - The Plant Journal - Wiley Online Library
Apoplast vs cell wall is going be a challenge. You could use immunochemistry (eg. Western blots) of culture medium to detect a secreted protein. GFP tagging may or may not work for direct visualization of secreted proteins as the pH of the medium will impact fluorescence.
You could try wall cross-linking followed by isolation and blotting.
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I have recently started working with arabidopsis and every time I pour the agar plates, I start seeing contamination after 3rd or 4th day.
Usually, there is no contamination after I pour the agar and let it sit for one day.
The contamination occurs on some of the seedlings, as well as some random parts of the plate.
I try not to pass my hands from on top of the plates, I UV the hood and the plates for 30 mins, and always clean the hood with 70% ethanol before starting.
I am open to any suggestions on how to improve myself.
Thank you.
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The contamination could be from the endogenous pathogens on your seeds. You can avoid this by sterilising the seeds for a short time before sowing them on the plates. Wrapping the plates with parafilm helps. The contaminants can also be screened out by adding a plant-friendly fungicide or bactericide to your media before pouring.
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I would like to validate the results of RNAseq but I am not familiar with this. I currently have a table with Gene IDs (Ciclev10004574m.g for example) and their Arabidopsis homologs (AT5G52300.1 for example) and expression levels. I selected the DEGs based on p value and log2FoldChange. However, for the design of primers, I don't know if I should use the gene sequences from my species of interest or their homologs from arabidopsis? or are they the same thing?
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For my own project, I had Arabidopsis homologs as well as geneID from species of interest. And for validation of candidate genes using RT-qPCR, I used sequence information from the species of interest rather than Arabidopsis orthologue. I hope it helps.
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Please give suitable suggestion for the study of FISH?
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Follow the work of Martin Lysak
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Hi,
I was asked to help to set up an arabidopsis facility as I was working with arabidopsis during my PhD and the person responsible had a serious accident.
During my PhD we had a green-house technicians who delivered pots ready to sow the seeds, so I have no idea what company was the soil:vermiculate/perlite mix.
Could you please recommend me some? They are planning to do some soil microbiome - plant interaction in the future, so the rings will not be suitable.
I will be very gratefull for any help,
Dorota
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Saveer Biotech company
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So I have always used Hygromycin/Kanamycin selection for Arabidopsis but this is my first time I am using methotrexate for selection of T0 transformed plants. Plants with hygromycin resistance I generally keep in dark and in3-4 days the one with resistance grows well.
I found only 2-3 papers where methotrexate has been used and it was not clearly mentioned that if there are more conditions for methotrexate selection other than just ading methotrexate to MS medium.
The working concentration I am using is 0.1mg/L.
Please let me know if you have experience with Methotrexate selection.
Thanks in advance.
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Hi there, I've used MTX regularly and have created several mutagenic Arabidopsis lines using this selection. It's very effective and easy to use. Just place it in the medium at a final concentration of 100 ng / mL* and sterilise / plate your seeds from transformed plants as you normally would.
MTX affects root growth by inhibiting the function of Dihydrofolate reductase, part of folate metabolism pathway, and the resistance gene is a variant form of the DHFR gene that is resistant. So sensitive seedlings will germinate and will start to grow a shoot and root, but will stop when the root is ~1 mm long. Resistant individuals will produce a healthy full-length root - quite easy to find in the plate.
* this is 0.1 mg / L as you say: to get to this low concentration, I dissolve the MTX powder in DMSO to a final concentration of 10 mg / mL (should be strongly yellow), then make a working stock by a 1:100 dilution to 100 ug / mL (weakly yellow). Then use this working stock at a 1:1000 dilution in the media (so 50 uL in 50 mL media) to final concentration of 100 ng / mL. Make the stocks fresh, it doesn't seem to work so well when it's been frozen / thawed. Good luck!
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I work in an industry so can't use Addgene.
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If possible you can try at Arabidopsis thaliana amiRNA library
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Hi,
I'm moving from Arabidopsis word to that of soybean, and I wonder from where one orders seeds.
In particular, I'm looking for the common Williams 82 strain.
Can anyone help e with providing a link or sharing some seeds?
Thank you,
Yulia
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ICAR-IISR, indian institute for soyabean research, indor, India can help you out
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According to the description on TAIR, the vector inserted in WiscDsLoxHs mutants (Arabidopsis) is pDsLoxHs. However, I cannot find the sequence of this vector, I am asking for help.
Thanks.
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You can check this article: J Plant Res (2007) 120:157–165 DOI 10.1007/s10265-006-0048-x. The WiscDsLox T-DNA collection: an arabidopsis community resource generated by using an improved high-throughput T-DNA sequencing pipeline
There are two T-DNA specific primers you can use (in above article): T-DNA-specific primer LT6 (5'-AATAGCCTTTACTTGAGTTGGCGTAAAAG);
T-DNA-specific primer p745 (5'-AACGTCCGCAATGTGTTATTAAGTTGTC).
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Dear All,
I am trying to generate over-expressing transgenic lines in Arabidopsis for my gene of interest by using Agrobacterium-mediated transformation. The protocol I followed for the floral dip was Steven J. Clough, Andrew F. Bent (Floral dip: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana).
However, no silique formation was observed in the transformed plants. I have repeated the experiment three times with around 30 plants but no silique formation was observed. However, the flowering was normal. What can be possible reasons for no silique formation?
Also, I have even tried to grow the deletion mutant lines ordered from NASC, even those mutant lines didn't even germinate.
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Hmm, it is more likely to be the dip experience for your plants than you gene interfering with all pollen grains. Dipping plants in soapy bacteria water is stressful. You could try and parse out the issue by testing leaving out all but one ingredient (silwet, sugar, Agro) then seeing how your plants do. I had a colleague with dip issues and their plants were getting heat/light stressed as they put humidity domes on their plants then left them in the growth room instead of in a low light environment for the overnight humidity time.
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I am new in the field of plant nutrition and I am searching for the most relevant markers to use to follow N and P starvation in Arabidopsis (both in shoots and roots). What are the most sensitive markers and those that would NOT be modulated by other signals (like microbial signals for example)? I thank you in advance for your help.
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Please consider chemical analysis for acid extractable phosphate and for a suitable form of mineral N related o what you are supplying, e.g., nitrate
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Respected researchers,
I am doing some in silico analysis on Arabidopsis thialiana's SOG1 protein, is there any 3-D structure, please recommend it, although I search a lot but could not find it.
If not, please let me know which tool or software will be reliable for creating the said structure.
Thanks
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Maybe with AlphaFold now you can get a better result
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Can anyone suggest me how to avoid fungal infection on regeneration media while doing agrobacterium mediated transformation of arabidopsis leaves by co culture method?
For agro removal i prefer to use cefotaxime and that works well but i immediately get fungal contamination hence no further callus initiation. This has happened several times with me in case of tobacco and arabidopsis both.
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I have had this in my explants as well. It looks like a similar fungi. I have found that it is in the soil of my plants and the spores must be on my explants and hard to kill with surface sterilization. My next plan is to empty the soil, wash the roots and transplant into fresh soil after I sterilize my plants. I also use PPM 1ml/L in my media to help reduce fungal contamination. Fungizone can also be used in small concentrations, but I have not researched that concentration yet.
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Hi all,
I am currently performing the bioluminescence imaging and fluorescence imaging in Arabidopsis root epidermis. As I used a signal peptide to secrete the fluorescent protein outside cells, by which I concluded it was localized in the apoplastic fluid. Even though I was able to confirm the presence in the apoplastic fluid (with the detection in the extracellular space of plasmolyzed cells), however, for apoplastic fluid specific localization, I want to check if my protein diffuses outside the apoplast hence localized in the cell wall matrix or accumulate on the cell wall surface. Is there any methods to confirm that?
If anyone has any suggestion or answer, I would very much appreciate it!
Sincerely,
Quang
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بطريقهSDS
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Are there any tips for obtaining clear images (20x) of cell files in each zone of Arabidopsis roots?
I normally fix them first in 96% ethanol and mount them in chloral hydrate. However, due to the ethanol, the roots are wrinkled, starting at the elongation zone (the meristem is okay).
Reducing the timing of ethanol clearing does not alter this effect, nor does using a lower percentage of ethanol, as observed in my samples with GUS-staining which are conserved at 70%.
I could try increasing the percentage, but this will be time consuming to apply to each sample.
Does anyone have any other ideas?
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You may use visikol. Put the seedling in a slide, add few drops of visikol, put the cover slip and incubate for 10 minutes at 37C. Your root cells will be cleared like magic. We routinely use this in our lab. Previously we used to use conventional cell clearing method using acid-alkali and alcohol. You can find that method in Malay and Benfey Development paper on Lateral root. This method also works well but takes longer time. Visikol is little expensive but works like magic and shorten the processing time.
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Dear researchers,
I used Agrobacterium tumefaciens to transform Arabidopsis thaliana, and the vectors were constructed on pCAMBIA1301 and pCAMBIA1302 with a hygromycin resistance gene on them.
I have harvested T0 seeds, and planted them on a selective medium (MS agar + 50mg/L hygromycin B), but after more than a week, all the seedlings looked the same as the negative control (col-0). (Attach pictures)
Even though I repeated the transformation experiment and harvested the seeds (To), used freshly prepared MS medium, and used freshly prepared hygromycin (bought a new one),
So even the second time the results were the same as the first time, all the seedlings looked the same as the negative control (col-0).
Has anyone encountered this kind of problem?
What should I do when I choose existing transformed seedlings?
Thank you very much.
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I was refering to a positive control
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Hey everyone, I just need some help with finding homologs for an organisms I am research. These would be the ADP Glucose Pyrophosporylase gene and the Glucan Water Dekinase genes, I have obtained the Arabidopsis sequences for the genes and now I want to use the Homolog function on Legume IP V3 to find the related genes in Pigeon peas (Cajanus cajan).
I have been trying to change my parameters but nothing seems to help.
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How can I calculate the relative expression in a transgenic plant?
I transferred a gene to Arabidopsis through the floral dip method (this gene is not present in Arabidopsis). I studied the expression of this gene in real-time. Can I use one of the LIVAK equations for it?
My data includes the reference gene and the target gene. I know that one of the lines with the lowest expression and the highest CT can be considered as a calibrator, but it doesn't make any sense to me, because treated and non-treated are in fact the same, what is your suggestion?
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I am not sure if this paper helps
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I have two arabidopsis mutants of SALK and Wisconsin Lox. I genotyped them with LP-RP primers and the LBB and P745 primers to check the homozygous lines. When I've isolated the RNA from it and used the cDNA to run a PCR with the same LP RP primers, I'm getting an amplification of two bands in wild type columbia and a single band in all the TDNA mutants. Can anyone help me figuring out the results?
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Amy Klocko Thank you for the tip! Highly appreciate it.
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Hi everybody,
we performed a proteomic analysis on dexamethasone-induced Arabidopsis plants, for 6 hours, and we verified with WB that our induced protein changed intensity (about 4 times). Unfortunately, the label-free proteomic of the same sample did not show any difference for this protein. The intensity found was not too high to suspect a saturation of the signal (moreover, we repeated the analysis using diluted samples but nothing changed). Protein considered was not imputed.
Could be a digestion a problem? We digested 200 ug of proteins with the FASP digestion method.
Any of you have experienced a similar problem?
Any of you have performed a label-free proteomic analysis after a dexamethasone short gene induction?
Thank you!
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The protein was identified and quantifed with 13 unique peptides. I did not observe big differences in peptides amounts in each sample..
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Is BRASSINOSTEROID INSENSITIVE 1-Associated receptor Kinase 1 (BAK1) a plant resistance (R) gene?
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Brasinosteroids, defined as the sixth plant hormone after the classic plant hormones auxin, gibberellins, cytokinin, abscisic acid and ethylene, are analogous to animal steroid hormones in structure. BR-insensitive (BRI) is the receptor for BR. BRI1-associated kinase 1 (BAK1) is another enzyme.
BRI1 and BAK1 interacted with each other in vitro and in vivo, which contributed to BR signalling 
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I am doing a phylogenetic analyses of two sets of proteins (A and B) that are functionally very closely related and share a "large degree" of sequence similarity. I have identified from various species protein sequences I want to include in the analyses (for both proteins). Each separate sequence was included based on their similarity / BLAST results to the known and characterized (functionally) proteins (A and B) in Arabidopsis. I am worried that some of the species included might however represent paralogs and not orthologs. Is there any analyses where I can "plug and play" the data that I have and see whether it comes out as orthologs (hypothetically then an orthologous group for protein A and one for protein B). I do not want to do an analyses where I search a database for orthologs, I want to ID it in the sequences I already have in my dataset (which were included obviously based on certain pre selected criteria). Not all the species / sequences we include might be from completed sequenced and annotated genomes.
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Hi Rouvay,
If I understand this question correctly, one way to proceed could be to evaluate, by mutual alignments of your n protein datasets, on your n assembled genomes.
Synteny could possibly be used to identify/differentiate orthologs from paralogs. However, tandem duplication could be a problem...
Another way could be to use dedicated tools: orthomcl, OMA...
Romain.
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Dear colleagues, fellow scientists and students,
I am working on establishing of protocol for BioID in plants. I am aware of already existing protocols such as cell culture, the transient transformation of tobaccos and root culture. For our purposes, we will need to work with seedlings and I was wondering if someone ever tried to perhaps transfer the seedling onto solid MS media enhanced with biotin. Could that work? What would be a good concentration to start with? I'd be grateful for any insights or links to relevant literature backing up the yes-no argument.
Thank you and all the best in your research!
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Tomás María Tessi Thank you for your input!
That is exactly what we wanted to do initially however we need to do a treatment to plants that should be 24-48h and I do not think the plants will survive it. Or they would be so stressed that the whole experiment will be biased.
So I am thinking about alternative methods. But perhaps I can do the treatment and transfer them to the liquid MS+biotin as biotinylation itself is 2-6h.
Thank you and good luck in your research too, maybe I just needed to look at it from different angle :)
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I am searching for a well defined protocol for Arabidopsis growth in a growth chamber with respect to following things:
1) Optimum temperature, light, light-dark cycle, humidity
2) How plants could be grown pots with potting mix? (Any requirement of covering of pots, trays? Watering methods/requirements etc.?)
etc.
Anyone to help with this?
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Hi,
Here you can find a well described protocol for soil and growth medium recipes. Also protocols for seed sterilization. https://bio-protocol.org/bio101/e126
Regarding the growth conditions depends on your interest but 60–100 μmol m–2s–1 of cool white fluorescent light, temperature 22°C and 50-60% humidity is our standard. For the photoperiod you could use 8 - 16h (light-dark), 12-12h or 16-8h, short-day, neutral-day or long-day, respectively. It's up to you and whatever you look for as the phenotype of your plants will vary a lot depending on the photoperiod.
Cheers!
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Good morning to everybody, I am working on article with tobacco and Arabidopsis genomic data and. Does anybody know database, server, or programm where I could find orthologs of Arabidopsis thaliana genes (e.g. At5g05630) in Nicotiana tabacum genome (e.g. Nitab4.5_0001047g0100)?
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Can the hypersensitive response occur as part of PAMP-Triggered Immunity (PTI) as well as Effector-Triggered Immunity (ETI)?
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Dear @Ruby Metzner
The ubiquity of hypersensitive response among higher plants despite its costs suggests that it is an extremely effective component of the plant immune system. Please check the following links, and attached pdfs; hope, these could be useful to you.
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Hi everyone,
I have been struggling with a PCR amplification for weeks now. First, let me explain to you the context:
I'm amplyfing a gene for a subsequent cloning (the gene is 2.5kb, arabidopsis)
I'm using a Phusion ploymerase.
Now here comes the incredible thing.
The first 4 PCR amplified my gene perfectly, but I needed to do more. From then, I always get a terrible smear (the one in the picture)
I tried EVERYTHING.
New primers, new reagents, new cDNA, different concentration, 2 different buffers (HF and GC), different temperatures, different PCR machines.
NOTHING seems to work, but why it did in the first place?!
Has this ever happened to some of you? What could I do?
Thanks for the support
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Which one of these samples is the no template control?
This looks like you have post pcr contamination in your assay and everything is reamplifying the contamination so you have huge over amplification. Try running a pcr with no added dna and see if you get the same result
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I am looking for a gene sequence in wheat that is identified in Arabidopsis, maize, and rice. I have tried to blast that sequences in wheat genomes (URGI, Ensamble plants). I did not get that sequence. I will be thankful if anyone can guide me.
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Hi. I hope the following article might help:
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Dear all,
My question is related to the nomenclature for naming Arabidopsis thaliana alleles.
If I understood correctly, for the moment, the convention in Arabidopsis for naming a new allele is to associate a number with the name of the gene, increasing this number compared to alleles already published (for example, if the alleles xxx2.1, xxx2.2 and xxx2.3 are already published, I have to name my new allele / mutant xxx2.4). Is there another mutant nomenclature used for Arabidopsis thaliana which more precisely identifies the type of mutation we are dealing with (e.g.: deletion in the promoter / in an intron / in an exon…)? For example, a bit like what is apparently done for the human genome according to this source https://varnomen.hgvs.org/
I ask this question because I have the feeling that today everyone wants to mutate their favorite gene with the CRISPR / Cas9 technique, making it possible to obtain and identify a new mutant more quickly. It also means generating a plethora of different alleles for the same gene in different labs simultaneously. So, I was wondering, if we continue to respect the current convention, if in a certain time there could not be more and more different alleles published with the same name?
Thank you in advance for your answers!
Cheers!
Marjorie
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May be you can reserve the name by contacting Araport?
I know that for new gene symbols you can reserve the name before register. Of course you need to check in the oficial list if the name exist or not, but you need to contact Araport anyway to register name, so I think they might tell you if the name is available or not?
:)
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Hello all,
I have an Arabidopsis mutant. When I grow it, I found there are lots of white spots on the leaves. I know some insects, like thrips, will feed leaves and leave scars. But I didn't find a clear insect on the plants. So I doubt it's the intrinsic reason. Could anyone help share some thoughts on this phenotype?
Thanks very much.
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Dear @Xian-Hai Zhao Arabidopsis belongs to Brassica family. Various kinds of leaf spot diseases including rust caused by different fungal species have been reported in rapeseed - mustard. You better know about the environmental conditions in which the mutant has been growing. I suspect it may be a fungal disease.
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The Arabidopsis wild type plants transformed with the transporter gene construct (gene + PBI121) has given me the T0 seeds. The T0 seedlings selected with kanamycin (50 mg/L) when placed in soil, grow up to the silique stage after which the plants are getting wilted and eventually die. Anyone could help me with this?
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Dear @Suhas Karle Whether all your T1 plants got wilted at siliquae stage? If so, then there is a greater possibility of management and diseases issues. Even to rule out all these possibilities, you should have grown the parental type as the check!
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Hello all,
I have been trying to transform a gene of interest (Arabidopsis gene, in pMDC107 and 163) into Agrobacteria strain C58C1 for quite some time.
One construct has a promoter region of 200bp upstream of the gene, the second has a region of 2000bp upstream of the gene.
I transformed the 200 construct into C58C1 by electroporation without any problems, but the 2000 construct cannot be transformed partou!
I have also tried GV3101, chemically competent cells, triparental mating, etc.
Does anyone maybe have another idea/method I could try?
Thank you very much!
Best
Judith
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Hi. I agree with Judith .
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Are resistance (R) genes expressed in response to pathogen attack - or only defense genes - as part of the plant immune response? R proteins act as receptors to recognize pathogen effectors and an interaction between R protein and effector stimulates Effector Triggered Immunity (ETI) which itself involves the expression of defense related genes. However, are R genes also expressed as part of ETI?
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Dear Ruby Metzner Plants have developed a complex defense system against diverse pests and pathogens. Once pathogens overcome mechanical barriers to infection, plant receptors initiate signaling pathways driving the expression of defense response genes. I have attached some PDFs; hope these will provide useful insight regarding answer to your question.
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Hi everyone!
I have a few lines of a particular gene of which the earlier (T2 ratio) are not known. I want to do antibiotic selection of these seeds and I want to know which plants of next generation (T3) are going to be homozygous. Is there an easy way to confirm number of T-DNA insertion of my gene present in genome of individual plants? Like some PCR strategy.
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I used a 3 primer method for identifying the insertion of my gene in Brachypodium T-DNA mutants , the protocol was given on the T-DNA mutant website.
They must have a protocol for Arabidopsis on the TAIR or SALK website, did you check it?
Briefly it mentions using your Gene specific primer with the forward or reverse T-DNA primer. In one reaction you add both T-DNA forward+ reverse+ your Gene specific primer. I have attached the protocol for your reference, it worked perfectly after the PCR on the gel you will see one band for homozygous insertion and two bands for heterozygous insertion.
Hope it answers the question, feel free to ask any further questions.
Best of luck Avinash Sharma
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I want to generate stable Arabidopsis transgenic lines expressing my protein of interest fused with GFP/YFP under the control of its native promoter. I want this for localization studies. Please suggest suitable vectors.
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thanks everyone for your kind help
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Looking for Arabidopsis seeds WT for establishment of genetic transformation and genes charcterization.
Hig appreciation if you can provide it.
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Hi all. I just found a gene that doesn't contain intron in Arabidopsis and Brassica order. However, in other species, like poplar and rice, or even pine, all of them have 4 introns. Actually, it looks like all the other plant species have 4 introns. These introns just disappear in Brassica. Don't know why?
Thanks.
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Dear @Xian-Hai Zhao
Genes without introns are a characteristic feature of prokaryotes, but there are still a number of intronless genes in eukaryotes. To study these eukaryotic genes that have prokaryotic architecture could help to understand the evolutionary patterns of related genes and genomes.
You please also go through the below mentioned links; Hope it could provide further insight to your quest:
Best wishes, AKC
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I have visualized the alteration of the cell wall during drought stress with the confocal microscope. Now, I want to measure the cell size at different time points. Is there anyone who knows how many cells I should measure?
I have studied Arabidopsis at 7 time points (days 0,1,2,4,7,15)
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Dear @Maryam Zekri The number and size of samples matter the most. Taking large size and more number of samples will greatly reduce "bias" from statistics applied. Moreover, I do agree with the suggestions given by @J.D. Franco-Navarro.
Best wishes, AKC
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After I dipped seeds into suspension of potential plant growth promoting bacterias,this bacterias grown on the 1/2MS plates and then covered the seeds resulted seeds can't grew.
So I want to consult how to inoculates bacterias to arabidopsis efficiently and evaluates this plant growth promoting properties accurately.
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Dear Youbing Wang I have attached three PDFs; hope it will be useful to you.
Best wishes, AKC
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I have used 0.25%NaClO 30s, 70% ethanol 30s and then wash with sterilized water twice. But in this way, it is very easy to damage the leaves. The Arabidopsis were growing in a sterile environment, they are quite small and weak. Do you have any suggestions?
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I am starting to work in genome wide association studies (GWAS) in Arabidopsis and I would like to know what is the difference between GWAPP Web application and performing the analysis on R. Can I get the same results with either of these tools?
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Dear,
The web tool is also a better option but using R is best, as it offers a number of packages and tools that can be used according to the requirement
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I need to store long-time Ara seeds, then I would like to know how long can I keep them alive in freezer.
Thanks
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Thanks everyone for useful advice
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Hi, I am trying to culture shoots from roots in wild type Arabidopsis. I use 4-6 day old roots and treat with NAA (24 hours) for lateral root primordia induction followed by incubating in high concentration of cytokinin (25 microM) for direct shoot formation from lateral root primordia (As reported in recent publications- Chatfield et al. ‎2013; Rosspopoff et al. ‎2017). But I repeatedly failed to make shoots from lateral root primordia. No shoots are made even at higher concentration of 2ip or different cytokinin. Surprisingly I do get new initiation of lateral roots even at high concentration of cytokinin. Can anyone suggest me the reason.
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I would like to know the quantity used. (can you really use 2i?it may be different?)
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Plant beneficial microbes can boost plant immunity and growth.
They are usually applied to young plants, but I want to apply them to seeds.
With tomato seeds its easy to apply fungal spores to the seeds. Vortex. Dry. Plant
With small Arabidopsis seeds (col 0) , its a bit more tricky. They are smaller, have less surface area and weigh around 20mg for 1000 seeds.
What techniques have you used to treat Arabidopsis seeds or other small seeds with microbial agents?
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Description below may be useful:
i. Seed roll technique: Seeds may be rolled on 7-10 days old culture of fungi on the culture Petri plate, and Petri plate rotated in both clockwise and anticlockwise so that seed surface get covered by spore. The pathogen coated seeds are kept for germination on blotter paper, and then are kept in an incubator at 28- 30oC for about ten days.
ii. Seed soaking technique: This method is followed as paper towel technique. The fungus is cultured on Potato Dextrose Broth (PDB). Twenty ml of PDB is poured into 100 ml conical flasks and sterilized. The flasks are then inoculated and incubated for around ten days. The mycelial mat from the flask is removed and macerated in a warring blender along with distilled water for a minute. The inoculum is later collected in a beaker. In the meantime, seeds are immersed completely in the inoculum. These seeds are then placed side by side on a blotter paper (45cmx25cm with one fold) and are folded. The folded blotter papers are then placed in trays, and kept in an incubator at 28-30oC for around ten days.
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Are pathogenesis-related proteins and antimicrobial peptides encoded by plant resistance genes (R genes)?
I'm unsure if the genes for these proteins/peptides are types of R genes?
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I want seeds of Arabidopsis halleri. I tried to search NACS but i could not find.
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Dear Researchers,
I have Transcriptome/Genome data and want to list all the member of a KCS/CER gene family whose Domain AP2/ERF, FAE1_CUT1, according to the Arabidopsis database, so is there any tool or online database which can use to perform a quick search for all members,
if have then please share with basic step by step guidelines,
Thanks
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Stephan Schie Thanks for your answer