Science topic

Tobacco - Science topic

A plant genus of the family SOLANACEAE. Members contain NICOTINE and other biologically active chemicals; its dried leaves are used for SMOKING.
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Tolin S. A. wrote that "PVY has a wide host range among the Solanaceae, including 70 species of Nicotiana and is also found in annual and perennial weeds in 15 or more plant families. " (Tolin, S. A. (2008). Tobacco Viruses. Encyclopedia of Virology, 60–67. doi:10.1016/b978-012374410-4.00575-6).
But, I cannot find the list of these 15 plant families and their reaction to the infection. Is there any species of Prunis among host plants for PVY?
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If it is so that PVY can infect plants of Polygonaceae, Brassicaceae, Asteraceae, Geraniaceae, Plantaginaceae without symptoms, we must have a vast reservour of infection around potato fields and in private gardens.
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I want to demonstrate a transcriptional regulation by the coinfiltration with Agrobacterium in N.benthamiana the TF and a promotor sequence. But I don't know which strain of Agro is better for that GV3101 or 3010?
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Thank you very much Dr Nick W Albert for clarifying my doubts and for the excellent tips. I will take them all into account.
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Hello guys, thank you for browsing my question.
In short
I used ++NEBuilder HiFi DNA Assembly Master Mix (NEB-E2621S) to assemble up to 5 fragments products (2058bp insertions to 10953bp vector) and it failed.
I got three false positive colonies in (3 fragments assembling) O.N, and through miniprep got the plasmid and the digestion plus PCR shows bad results.
And after 48h I got a lot of colonies that cannot even grow in starters with antibiotics.
Please guide me the right way to success, thank you in advance!
Long and detailed version:
First of all, my cassette, the target gene is toxic to the dh5a, so I also ordered NEB® 5-alpha F' I q Competent E. coli (High Efficiency) to do the transformation.
I ordered primers which are 25bp long, and I used proofreading enzyme to gain the PCR product.
I digest and get the vector, it is pCAMBIA0390, for agro-transformation in plants.
So I got the PCR products 50ul, I ran 5ul on gel and found there are unspecific bands, so I cleaned both the digested vector and PCR product by the miniprep cleaning kit, then I got a very low concentration (around 2-10 ng/ml). All my PCR products are within 1200bp. And one of the PCR fragment has a lot of GC which is harder to get the PCR product, so I added a GC enhancer.
The biggest insertion (including vector are 5 fragments) is 2058bp, the whole plasmid with my insertions is supposed to be 10953 bp.
I added the whole reaction system as 20ul, and vector:insert = 1:1 according to their concentration (ng/ul). The PCR program was 60 minutes in 50c since I have 5 fragments.
Then I used 2ul and 18ul assembled products to do heat shock transformation to NEB® 5-alpha F' I q Competent E. coli (High Efficiency), and left the plates in several dilutions.
After 24h in 28c, there were 3 colonies appearing in  3 fragments assembling reactions. I grew the starters and did both digestion and PCR, no right products. The agar plates were kanamicin added, after 48h there were a lot of very small colonies that grew, and I tried to grow starter from them with kanamycin antibiotic, there were no colonies able to grow, the starters remained crystal clear.
So I was wondering if you know anything I did wrong or there is better condition for the assembling kit working (like digestion enzyme which acclaimed can work in 15min is always better to digest for 1h; and ligation which could be done within 0.5h but is it always better to do a 16h 16c.)
Thank you and looking forward to hearing from you.
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I do agree with all points commented by Mimi Asogwa and would just make a few questions to figure out what is happening.
1 - Why did your PCR product did not give you a one amplicon only? Did you try everything to optimize it or just followed some paper protocol? If you just followed someone else's protocol, then, optimize it and then use it. Not optimized reaction may lead to this kind of problem.
2 - Usually in all assembling reactions, again as Mimi Asogwa told you, it's better to use/try different vector/insert ratios and use a molecular calculator to find the most precise mass amount (in nanograms) for this purpose. So, a 1:1 ratio is not always the best (honestly, only when the sizes are very close to each other).
3 - Lastly, you could do it stepwise. First, take the fragments that makes your complete insert together and only then, a second reaction with it and the vector. Sometimes, it works better.
Hope my comments may help you.
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  • We expressed a maize CC-NBS-LRR protein in Nicotiana benthamiana by injecting the Agrobacterium strain GV3101 with a maize CNL protein into the leaves, cell death was observed after 3 days without the presence of the cognate pathogen effector, Does this happen often?
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Thanks a lot!
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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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The carcinogenic effect of smoking was finally proved in the 1960-/1970-ties. However, at that time DDT, Lindane and the like were sprayed onto the tobacco leaves, and the warming- and burning-products of these substances were inhaled by smokers.
Opposed to mice and rats used in trials, humans have been exposed to smoke from plant parts in thousands of generations. Humans are therefore likely to have evolved smoke resistance.
The eyelid could be a ”macro-example”. Has smoke resistance been shown in humans at a molecular level?
Please consider where your answer is most relevant. You can of course also answer both places.
This will be helpful to the readers of RG)
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Hello,
Yes, humans have evolved smoke resistance because humans carry a genetic mutation in the AhR gene that reduce our sensitivity to cancer causing chemicals found in wood smoke.
The AhR gene codes for aryl hydrocarbon receptor that helps regulate our response to carcinogenic polycyclic aromatic hydrocarbons often found in wood smoke.
The AhR is a ligand-activated transcription factor in eukaryotic cells that alters gene expression in response to a wide range of exogenous and endogenous molecules including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
AhR also regulates the function of proliferative factor E2F. TCDD activates physical interaction between AhR and pRb promoting binding to E2F and stops cell-cycle. Also, TCDD stimulates interaction between AhR and p300 which leads to displacement of p300 from E2F sites.
I hope this is helpful.
Best.
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Can I use LepR/LepF primer to amplify mtCO1 gene and determine biotypes of Bemisia tabaci?
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Yes, you can use.
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Hi everyone,
I have a very troublesome time when I trying to build the construct of my gene. Since the gene sequence of my target insert is toxic due to the leaky expression, I used the competent cell that ordered from NEB company: NEB® 5-alpha F' Iq Competent E. coli (High Efficiency) to reduce this problem, and I encountered trouble when I tried to extract the DNA from the starters.
I used traditional digestion to get my insert and the vector, the I did ligation. After the colonies grew on the ampicillin plats, I did colony PCR by the use of my insert primers, it shows the very good result that my insert is inside of the gene of the colonies' cell.
Then I used the colonies to grow the starters (O.N. in liquid LB+amp.). I also did the starter PCR, but in the comparison of the positive control (the plasmid where I get my insert from), which has strong right size band; the samples shows nothing.
After that, I redid everything, and miniprep (i.e., the DNA extraction). In addition, I digest the extracted plasmid. The intact plasmid looks 2kb smaller compared to what it should be, also the digested bands.
What could be the problem during the process? If the idea that using low-copy number e. coli is not really working, is there any other way that I can use to build the construct with this toxic gene? My aim is to transfer the gene into tobacco and produce protein.
Thank you so much and looking forward to hearing any constructive suggestions.
Best
Ruojin Tian
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I have seen these positive results before from colony PCRs when you use the colony directly from the transformation plate. If you are using primers inside your insert and a good DNA polymerase, you can amplify your insert from the fragments of DNA that are spread over the plate, not inside the cells. This DNA comes from the ligation you used to transform the cells and the colonies that you obtain contain a empty vector.
To solve the problem with the colony PCR, you should use primers located in the vector that produce amplification of your fragment. If the fragment is really long, you can use a primer inside the insert and other in the vector.
Concerning the toxic expression of your insert, do you know if the expression is due to your vector or is your insert which is been recognized by the transcription machinery?
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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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First picture is vector construction for express eGFP by using Agroinfiltration.
But no fluorescence was detected. So what was the problem.?
I used A. tumefaciens GV3101 strain and pGWB505 Binary gateway vector, and nicotiana benthamiana for inoculate plant.
And I observed inoculated leaf 7days post inoculation.
I guess the vector construction is wrong..
Please give me the advices.
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I am using pGWB405 vector and am not able to detect fluorescence either. However, I did RT-PCR on the infiltrated leaves, I do see RNA expression. Also, I've realized that 7 days is too late. I am doing a time course experiment to track the expression.
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My question is as the title.
I found that I cannot find an exact explanation on what are the bands that I see on this gel. I would suppose some of them are rRNAs, but which one is which?
From what I searched, there are four rRNAs for tobacco leaf, but why I have five bands?
Thank you!
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You must have contamination in your RNA extraction if you see an extra band on the agarose gel.
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I am looking for few common tobacco pathogens as well as other general plant fungal pathogens. If anyone will be willing to provide it then please contact me. 
Thanks
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Lot of work has been done by Tobacco Research Centre at Nipani, Dist-Belgavi, Karnatak state(India). You may contact Director of the Research centre .
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Hello,
for my research I was wondering on the fate of glucose if I were to agroinfiltrate a high concentration of Glucose (eg 30mM) into the plant:
  1.  will the glucose be translocated to sinks? I'm aware that normally it does not necessarily get shuttled as the "pure" monosaccharide form.
  2. If yes, does this only happen at night? Or are there other mechanisms that can operate to regulate the concentration?
Thanks in advance.
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Chinaza Godswill Awuchi thank you kindly but neither of these links give me the answer as to the fate of the glucose I were to apply. Indeed, they focus on the fate of the bacteria agroinfiltrated, rather than the fate of the glucose.
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Hi I am trying to express my protein R in tobacco leaves and do a CoIP, but I can't detect the protein R in CoIP input.
I took the same tissue and added 8M Urea plus LDS protein buffer, protein R shows as a distinct band at the correct size. But when I grind the tissue and put in CoIP lysis buffer, after centrifugation, I put the input in LDS, I couldn't detect my protein, I only see a smear.
Does anyone know what went wrong?
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ok, we´re working with the Plant Protease Inhibitor cocktail from Sigma (P9599).
This works also fine.
Best and good luck furthermore!
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We know very well that RNA silencing is a potential ways by which small plants such as tomato, tobacco etc protect themselves against viruses. I was just curious to know if RNA silencing is also equally effective against virus infection in big trees? Although, I am also not sure if viruses infect big trees.
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RNAi is also effective in trees but it depends upon the time and way to transform and RNAi inductions. The expression will persist or not till the maturity levels, is also another source of concern.
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Though tobacco is considered to have high levels of toxic compounds, nicotine being the most abundant, from my observations those who chew tobacco have up to 99% not suffering from dental problems. Comparatively with smoking tobacco those who chew are not at risk of lung cancer, or throat cancer.
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Good Answer Joe Graymer
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Hi all,
I am planning a research project for next summer that investigates the effects of high temperature exposure for different lengths of time on the percent nicotine concentration in tobacco leaves. The setup is as follows:
Tobacco growing conditions
3 experimental groups of 10 or more will be grown in a chamber with a peak daytime temperature of 35oC and a minimum nighttime temperature of 26oC.
  • group 1: 3 days exposure to heat
  • group 2: 5 days exposure to heat
  • group 3: 7 days exposure to heat.
3 control groups of 10 or more plants will be grown in a chamber with a peak daytime temperature of 30oC and a minimum nighttime temperature of 21oC.
  • group 1: remove after 3 days
  • group 2 remove after 5 days
  • group 3 remove after 7 days
When the plants are removed from the temperature controlled growth chamber, I will dry the leaves in a 40 c oven until they are dry enough to powder. I plan to extract nicotine from leaf powder with methanol in an ultrasonic bath. Once I filter the resulting solution to remove particulate matter, I need to find percent nicotine. My school has a Hewlett-Packard GC-MS (NSF 9851032) with an autosampler. I am an undergrad with more background in bio than chem, and I am unfamiliar with gc-ms. Could I use this equipment to quantify nicotine in the leaves, or does this equipment only work for identification? If I can use this equipment for quantification, what general procedures should I follow? Any additional advice on methods would be much appreciated as well.
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Use a nicotine (alkaloid) selective, or a general extraction procedure, concentrate your extracts, weigh them all carefully, prepare a sample of known concentration (mg/ml) for injection, serially dilute to required levels of concentration (check GC-MS analysis of nicotine procedure, sample pre and injected micro litre sample quantity), the weight of the extract in the injectable volume is a must here! Run your sample, if need be use spiking techniques, record GC and (GC-MS comes later, MS another confirmation for Nicotine, but just don't rely on this info only, compare and see in known nicotine spiking to the injected sample!), identify the nicotine peak from the known column, concentration, and RI (not the RT- Retention Time) values, so the spiking for nicotine confirmation is necessary. Get total peak are, nicotine peak are, calculate the peak % for the nicotine, and correlate with your sample quantities. Repeat for all extracts, and get to know your concentration of the nicotine in the sample - each extract! Precaution: record all weight, volumes, procedure in stepwise manner, including GC-Conditions!
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How can I extract nicotine in good yield from tobacco (and the good way for determination of it in extraction)?
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The following link describes a simplified way to extract nicotine.
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I'm writing about a type of smokeless tobacco used in asia (nicotiana rustica linn), my purpose is to find out about heavy metals such Cd, Ni, Zn, Cu and etc in nicotiana rustica linn.
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Adekunle Adeleke, thank you for your good advice. nicotiana rustica linn is one of our most popular smokeless tobacco products and is widely used in the southeast of Turkey. there are many studies that have been made about how it affects health and economy. but still no studies on major heavy metal in nicotiana rustica linn have been presented. to the community. I am preparing this research for postgraduate degree purpose, so I wanted to share it publicly in order to gain knowledge, skills and academic success.
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It is necessary to determine the activity of the enzyme glutathione-S-transferase in the roots of tobacco. Help me choose the most optimal technique.
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Please check this PDF that was uploaded by a colleague and I found it highly valuable and helped me.
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I want to know if anyone knows some statistical simulation model that relates tobacco with childhood asthma. Recently we had ​​a publication, but found very little information.
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Please also see the following PDF attachments.
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It is difficult to work on crops and chances of successful experiments are very less, that is why we prefer these model organisms to study the mechanism and then apply the same knowledge on other crops. So, according to you, for different traits and developmental studies which is better?
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I have worked with Nicotiana tabacum and Arabidopsis thaliana. As for me tobacco is more easy culture to grow and work with. But for arabidopsis there is full genome and special resourse about genes and etc https://www.arabidopsis.org/
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Thank you in advance.
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Please also have a look at the following PDF attachments.
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hello all,
I am trying to produce His-Human-TGFB1 in tobacco leaf tissues using agrobacterium mediated transient expression system. The Purification is based on His-Tag Ni-NTA system. Protein is being purified but 60-80% of eluted protein consists of plant protein.
Conditions are as follows:
Note:His-TGFB1 is hydrophobic in nature; a Dimer; commercially available protein is recommended to reconstitute in Citrate buffer pH=3.0 or 4mM HCL
1. extraction buffer: 50mM Tris or 50mM PBS (pH=6.0), 300mM NaCl, 0.2% tritonX100, 1XPI, 5% glycerol, 10mM Imidazole, No DTT.
2. washing buffer is same as above except No PI, DTT,Triton. Imidazole con 25mM.
3. Elution is in same buffer with 300mM imidazole and no PI, DTT, Triton etc
I am looking forward for the suggestions. All are welcomed.
Thanks in advance
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@Aditya Soni: In general, magnetic agarose beads should be preferred, when non-specific binding is an issue. In this field there are some suppliers, including Cube Biotech, which sells high capacity Ni-NTA MagBeads for a reasonable price. And, in addition to that, INDIGO Ni-MagBeads, which are tolerant against EDTA and DTT. There is a Korean distributor, too...
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As health care advocates for children, pediatric staff and family medicine have to alert parents and older children about the risk of potential nicotine use among children and teenagers.
Several tobacco promotions may reach children in early age, either through social media or peers.
Many smoking habits and other forms of nicotine use begin in adolescence, hence the vital role of the health care system to prevent and proactively addresse this risk before such health risk problems arise in this vulnerable population.
As health care advocates:
What's the best way to address nicotine prevention among children and parents?
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Greetings!!
Its a very important topic indeed. Nicotine/Tobacco usage especially among children and teenagers is on a rise, globally. Cancer is a global problem, and regular, indiscriminate consumption of tobacco and related products is thought to be a crucial factor for development of oral malignancies. A lot of factors play in the initial urge among the teenagers/children for using tobacco. It may be due to their curiosity; may be due to bad company of friends; may be due to attractiveness towards the flashy ads of various tobacco products in either television or internet; or may be due to less knowledge of the harmful effects due to long-term exposure. To curve this global menace, the solution has to be solved both at an individual level and as well as strong policies should be taken by the concerned government health agencies. Necessary educative course material regarding harmful implications should be introduced at school/college level to discourage tobacco usage. Practice of healthy lifestyle should be encouraged among the youth/children. Respective governments worldwide on their side can increase the taxes on tobacco related products, which might discourage its usage among the public.
As, you have said and i agree that ".... Several tobacco promotions may reach children in early age, either through social media or peers....". Yes, in this age of high speed internet connectivity this children are more addicted to their computers/mobiles and get inspired from the flashy ads/promos. To solve this regular internet ads, forums etc with a strong focus on chronic harmful effects will be a step towards curving this menace. Health advisory symbol/image in the packaging of all tobacco and its associated marketed products should be strongly implemented.
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Tobacco is a deadly habit which is very common among all societies yet what we want to ask ourselves is: Does imposing price increments limit the habits among the smokers ?
It is a fair question to ask!
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The WHO argues that increasing the price of tobacco product is the most potent and cost effective policy in tobacco control. The WHO insists that higher price of tobacco product would reduce the affordability of tobacco products. This, in turn, would induce consumer to cease smoking, while at the same time prevent others from start smoking. In agreement with Paul Stock Paul, it is deemed that price elasticity of demand for cigarettes is at average - 0.40 in high-income countries and about -0.50 in low- and middle-income countries. Thus, it is assumed that lower income people tend to be more sensitive to price change. In addition, among all taxes, tobacco excise is the most effective one as it is exclusively levied to tobacco product and it raises the prices of tobacco product relative to price indices. Accordingly the WHO urges member countries to (1) increase tobacco excise at least 70% of tobacco product retail price, (2) ensure that the increase of tobacco excise higher than the increase of price indices and income, (3) prevent consumer from shifting down to cheaper tobacco product, and (4) strengthen tobacco excise administration. The WHO also maintains that higher tobacco excise tax would provide governments with reliable stream of revenue which can be allocated for improving public health and strengthening tobacco excise tax administration.
However, many researchers suggest that substantial tobacco excise increase would bring undesired impacts. Excessive tobacco would encourage tax evasion and avoidance as it offers large incentive for illicit production and trade of tobacco product. In line with Robert van Brederode, smokers would not reduce tobacco product consumption. Instead, they would shift down to illicit tobacco product which is certainly cheaper. As a result, excessive tobacco excise would simultaneously undermine public health objectives and damage tax base. Policy makers, for that reason, carefully increase tobacco excise while concurrently keep illicit tobacco product under control.
Please find more information on:
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can someone educate me on this one. thanks
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Hi,
there is no generic response to this question, as this will depend on the specific promoter and also the used fragment. E.g. the Ubiquitin1 promoter from maize was reported to function in tobacco (I am quite sure), although I don't remember the precise reference. But others might not, or they might lose their specific properties, as expression domain, strength, whatsoever. So if the promoter you are thinking of (you do not specify) was not previously tested, you simply can't know.
Anyways, is there any reason that prevents you from using a previously characterized promoter fragment, besides the effort of making the construct? Since it takes several months to make and characterize the transgenic tobacco, I would rather go for such a chimeric construct.
Bests,
Johannes
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I'm trying to transfer my construct into plant (Nicotiana benthamiana) using Agrobacterium LBA 4044 it grow at the first few week good, but when transfer my explant into selection media with Kanamycin 100mg/L and Timentin320mg/L the edge of explants turn brown dose that mean the Antibiotics very high in concentration.? do know how can I solve this please?
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Explants turning brown is totally due to over exposure to disinfectants. Browning is due to release of phenolic from the cut ends into the medium. To solve the problem change the R2 medium to 2 mg 2,4-D L- 1 and 20 g maltose L - 1 and they are carefully removed from dying explant tissue and transferred to fresh medium containing 50 mg hyg B L - 1 for further selection. For details consult https://books.google.com.ng --books.
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By 2040, according to a new report by the Institute for Health Metrics and Evaluation, the Spanish are expected to have an average lifespan of 85.8 years, outliving even the Japanese, who have long headed the global longevity tables. And outliving those of us in the UK by almost 2.5 years.
Does anyone have a suggestion why this might be in a population noted for its consumption of alcohol and tobacco?
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Dear , Dr. Anastas Ivanov Ivanov... Olive oil and fish may be reasons of long life , but the red wine as I think is not a raeson of long life, versa, it may be a reason of short life.
With my greetings.
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The idea of transferring C4 traits into C3 crops is not new and dates back to early years of this century when Japan Tobacco was granted a US patent for generation of PEP-CK type of C4 cycle in rice. The increasing levels of difficulty in meeting the food and fuel demand however, is again stemming up interest regarding C4 rice. With access to advanced technologies and monetarily self-sufficient international collaborations (like C4 consortium), much of the ground work like molecular tool development, unraveling the genetic infrastructure, collection and study of available genetic variability has been done. But still can we say that we will have first C4 rice cultivars by the next decade or so? or is this required in fact? I mean our C4 cultivars are not operating at full efficiency so why take our cereals C4, instead can't we just silence the photorespiration
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Exactly. Silencing photo respiration will be much more effective as C4 plants are not efficient partitioner of dry matter. Moreover, plants have to spend lot more energy in photosynthesis. Increasing CO2 level is expected to give advantage to C3 plants in near future if the increase in photorespiration can be controlled in elevated temperature levels.
Further, C4 cycle is most efficient for plants where total biomass is economic rather than grains.@sudershan mishra
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Hello every one. I need help please!
I did transient expression in Nicotiana benthamiana using GV3101 Agrobactirum strain. The bacterial cultures which have GFP were co-infiltrated with equal amounts of an Agrobacterium suspension of a p19. After 5 days the plants were analysed by confocal microscopy. Unfortunately I couldn't find anything and there was no signal for GFP.
I did this two times now and it did not work at all.
Can anyone please give me any suggestion that might help.
Thank you
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Hi thanks for your answer. Yes I am sure about the vector. But I screened them after 5 days some of our lab members told me that GV3101 is usually screend after 5 days. Do you think I have to screen them in 2 days?
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dear all,
My project is about the specific gene expression in tobacco hairy root tissue and I used NtREL1 promoter to produce transgenic tobacco plants with the ability of root specific expression. But my tobacco plants have problem in regeneration after transformation with the expression construct that contain this promoter. would you please help me in this respect? I would be grateful if you let me know your opinion about this issue.
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Hi,
You can check for contentration of antibiotic used for tobacco plants. If u dont know the working concentration, then you have to proceed with sensitivity test for particular selection marker in tobacco..
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Hello,
I am overexpressing a plastial enzyme in tobacco, and recently I saw that the younger leaves of some plants are showing signs of chlorosis (see image).
Although I dont have a lot of experience growing tobacco plants (generally just to infiltrate with Agrobacterium), I am not sure this is a nutrient deficiency.
Any thoughs would be greatly appreciated.
Best regards,
Henrique
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Fe deficiency. Mg deficiency first appears on older leaves, not on the youngest ones.
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Dear all,
I used agroinfiltration method to express YFP-tagged protein and FLAG-tagged protein in tobacco leaves, and after infiltration I observed the leaves by microscope, and the result showed the YFP protein expressed in the tobacco leaves. Then I tried to extract nucleus protein by various methods, but Western Bolt results showed that the antibody did not catch the protein, so I am wondering if there are some good protocols to extract nucleus protein from tobacco leaves.
Best Regards.
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Thanks for your precious advice, Mr. Castro.
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Hello,
I am trying to purify a recombinant protein (~15kD) from Tobacco. I am using Agroinfiltration based expression system. Expression is high, but when it comes to purification, I could not get Protein of Interest rather more non-specific bands. I extract protein in extraction buffer (50mM Tris-Cl, 150mM NaCl, 20mM imidazol, 0.1% tritonX100, 1mM DTT, protease inhibitor), and after getting soluble protein, it is passed through the Ni-NTA column and afterwards washed in (PBS, 30mM imidazole, Protease inhibitor). Final elution is done in PBS+250mM imidazole.
Please suggest me some strategy based on your experience. If someone is expert in this technique, their cooperation will be highly appreciated.
Thanks in Advance
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Thanks for the answers. Mr Shahed, could you please let me know the composition of the "Charge Buffer" and con of imidazole in EB.
Thanks in advance
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Some time ago we started to have some problems with our BY-2 cell suspension cultures. The cells started to flocculate and form consistent cell masses that are very difficult to pipette when subculturing. We already renewed the medium and components but the phenotype remains. We also tried to start again from callus culture, but we also get the same aggregates. Does anyone ever experienced similar problems?
Thank you in advance.
Best
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Anybody has the plant cell line tobacco BY-2 cells in USA?
I need this cell line for experiments. I am Berkeley, and I would be grateful if you could send me the line!
Contact me through ResearchGate!
Thank you!
Xiao
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Xiao, were you every able to find BY-2 cells in the USA?
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Does anyone know the molecular weight of component of N.glauca? which contain waxy and oil. I would appreciate it if someone could help to know the molecular weight of N.glauca. Thanks
Kind regards,
Peter
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Nicotine is ingredient of Tobacco/Cigarette-Tobacco. Is it possible to find nicotine and its compound in mixture of water and tobacco.  
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The term ‘‘tobacco constituent’’ is defined as a substance naturally present in tobacco. The term ‘‘tobacco ingredient’’ is defined as a substance (except water) that is added to tobacco during the manufacturing process and having a specific function on the final tobacco product. Tobacco ingredients are classified as flavours and additives. In 1960, a little over 200 chemical constituents had been identified in tobacco leaf of all types and less than 450 had been reported in smoke. Today, approximately 3000 have been identified and characterized in tobacco leaf and some 4000 in smoke. Estimates are that the total number of chemical constituents in leaf exceeds 4000 and there are over 6000 in tobacco smoke. Mixture tobacco for cigarette consist flue-cured tobacco- virginia, air-cured tobacco-burley, sun-cured tobacco. reconstituted tobacco (RECON) and expanded rib which differ according to the chemical composition. The smoke composition is depend from composition of tobacco mixture.
best regards
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Dear all,
Recently, I am working on the electrolyte leakage assay in tobacco with leaf discs. When the discs were dissected from the leaf and kept in a tube, I put them in -7°C (salted water) directly for 3 hours. Strange thing is the discs from the same leaf show different electrolyte leakage (some is 10% and some 80%). I do not know why this could happen. Does someone have some experience of working on that and some explanation?
Best,
Yong
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What part of the plant leaves are taken and which part of the leaves used for discs (you participate leaf veins on the disc)? Which electrolytes do you research?
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....
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thank you both of you #Ramsey Sir and #Nermina Madam
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We started preparing our manuscript on "Prevailing social obstacles in keeping tobacco-free homes in urban areas: Realizing ways to overcome challenges", which was fully funded form Institute of Global Tobacco Control (IGTC), USA  in 2016
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Hi. I think this paper could help with you manuscript preparation: 10.1017/jsc.2015.3 (Tobacco Cessation in Economically Disadvantaged Dominican Republic Communities: Who are the Ex-Users?). We used generalized linear mixed model to generate adjusted OR. 
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I want to identify factors associated with tobacco use among adolescents in a rural setting, and possible risk factors have been categorized into three: socio-demographic, behavioral, and environmental. Will it be right to create three models using hierarchical logistic regression so I can identify which model best predicts the outcome (tobacco initiation)?
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Thanks a lot John-Kare
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Why is tobacco rattle virus a choice in agroinfection. In spite of the virus being of bipartite RNA nature the gene of interest is introduced only in one of the RNA before agroinfection. why so?
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RNA-1 is capable of independent replication and systemic spread in plants, the others are not required and that is the reason only RNA-1 is introduced for agroinfection of plant hosts.
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I am trying to figure out how many Myzus persicae would infest a potato without top-down control (predator or parasitoid). In some papers, I have read that Myzus persicae is non-gregarious and therefore does not occur in a large number in a host plant. However, from my experience, a tobacco plant could hold quite a number of Myzus persicae and so could a chinese cabbage. Am I wrong to assume that there will be hundreds of aphids when there is no interference? Is Myzus persicae always gregarious or does it depend on the host plant it feeds on?
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Also from my experieince, hundrerds from Myzus persicae infested the sweet pepper, on which I reared huge number of M. persicae and these were protected from natural enemies either parasitoieds or preditors. I think it is gregariuos .
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i want to find questions to write in the questionnaire to find if there is a relation between lung cancer and tobacco smoking or not?
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Tobacco smoke is a poisonous concoction of carcinogenic, radicals, and intermediate by-products all of which are suspected to cause lung cancer. Research in literature is very elaborate on this. For your questionnaire, you will need a sample population of smokers, their smoking history, and their medical history. The surgeon general's report can come in handy when dealing with these sample population. A sample question to ask would be. "Ever since you started smoking, have you had any whizzing of the lungs during a cough?" etc. Good luck!
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I am not getting shoot formation from callus. I have used 2-8mg/l BAP and ten times less concentration of IAA. I found transformation in 3-5% of calluses. 
Only size of the callus increases in 16 hr light and eight hr dark periods. How could I solve this problem? 
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Dear Mohd Shadab
you can increase cytokinins concentration in the media at a certain concentration and used (BA and kintin ) together, with or without auxins , and you note response of callus on developement and regeneration of shoots.
must you separate the shoots carefully and thoroughly, must at all shoots containing on base when it cultured on rooting media and lenght appropriate
Thank you and Good luck
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Thank you
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Dear Mercy,
You may contact any genebank (Center for genetic resources) to get them. 
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Do you know the relation between the pharmaceutical industry and WHO efforts against smokeless tobacco? Are there initiatives against nicotine dependence or only against smokeless tobacco? I am afraid that focusing only on smokeless tobacco opens pharmaceutical industry to dominate the nicotine dependence market (by selling nicotine substitution products) without addressing the key of the problem. Do you know some analysis/publication in this field?
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You have said most. I agree.
Will human beings continue with a personally satisfying and, if properly controlled, relatively minor noxious habit (s)? Yes, I think so.
My original question just stressed the relevance of excluding market interest from public health. I hope no one finds the “Winerette” to avoid the glass of Rioja you may drink on Saturday evenings …
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We are trying to regenerate shoot from tobacco callus. for this purpose we have used different concentration of  cytokinin. 1-8 mg/l BAp,  kinetin, 1-8 mg/l BAP+kinetin and callus was put in 16 hour day and 8 hour night cycle but we didn't find any regeneration. callus become green and just increase in size.
then we use higher concentration of cytokinin that is BAP and very less concentration of auxin (1/4th conc. of cytokinin) that is 2,4-D. still we didn't get shoot regeneration. Conc. of BAP in this case is 2,6 and 8mg/l.
Is there any problem in the selection of phytohormones or there could be other reasons for plants were not regenerated.
kindly help me to solve this problem  
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Hi  Ali and thank you for your reply
pH of media always 5.6-5.8. and concentration of phytohormones are  as follow
experiment 1                         experiment 2                               experiment 3        
    BAP mg/l                    BAP+KINETIN mg/l                                  BAP+2,4D mg/l
              3                                   (2+2)                                                (3+0.75)mg/l
             6                                     (3+3)                                                (6+1.5)mg/l      
              8                                   (4+4)                                                (8+2)mg/l 
here we use 3 different concentration of phyto-hormones in 3 experiment. in experiment 1 we used only BAP in 2nd experiment we used kinetin with BAP and in 3rd experiment we used 2,4-D with BAP.
now we are using IAA with BAP and kinetin hoping to get some positive result.
if u now suggest me it will be valuable for me 
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is there any seed stock center for mutant tobacco? can anyone help me to find any?
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thank you so much ramsey. I will check them. 
regards
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Can anyone share information regarding procurement of authentic wild type Nicotiana tabacum seeds?
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There are two main sources of Nicotiana germplasm.  One is North Carolina State University
a second one is in Bergerac, France
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I'm search a laboratory assay applicable in field (like immunochromatography) to determining tobacco passive exposure by urinary cotinine, with a cutoff concentration of 1-2 ng/mL.
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Hi Fernando,
There are several papers from the ITC (International Tobacco Control) Collaboration which detail the technical means for establishing the physiological impact of passive smoking. The work was led by people from the Roswell Park Cancer centre in Buffalo NY and my advice is to contact Andy Hyland directly at that institution.
Good luck
Dave
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Genotoxicity of tobacco and alcohol is a well known fact. These are carcinogenic in nature, too. Is there any impact of these on germ cells of human  in general and determination of sex in particular ?
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This is a question for medics,
Best regards
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Stomatal aperture assays in abaxial epidermal peelings of tobacco leaves were done as described in Allen et al. (1999) and He et al. (2013). Peelings were induced to close all stomata in dark condition, and then a part of samples were kept in darkness, a part of samples were put in light condition to induce stomatal opening, and some samples were put in light with 50 µM of abscisic acid to induce stomatal closing (Schemes of the assay are in the following attached picture). Then I obtained microscopy images (10x, 20x and 40x) at three condition times (2 hours, 4h and 6h). I want to determinate the degree of stomatal aperture at those times in each treatment, but I don't know what kind of parameters I need to measure, and if there is any kind of equation that integrate those parameters. I imagine that important parameters can be: stomatal length and width, and stomatal pore length and with, but how can I end this story? 
Thank you very much indeed.
JD
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Some toxin like FC can increase to a max stomatal opening http://onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1974.tb02120.x/pdf. Opening is measured by pore area or ratio of width  to lenght and the value can be obtained by a variety of image analysis, Image J in https://www.colby.edu/academics_cs/courses/BI214/upload/lab5-stomata.pdf
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The digestion methods for plant materials are many. Which method may be the best
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Hi Dear Dr. Barbooti
Please read our article entitled: Comparison of the Level of Cadmium and Lead between the Cigarette  
Filters of Different Iranian and non-Iranian Brands" which we determined the heavy metal contents by Atomic Absorption Spectrophotometer.
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I am a research student at haldia institute of technology (Haldia, east Medinipur, west Bengal, India). I would like to research on tobacco plant (Nicotiana tabacum). So I need tobacco seeds. Can anyone help me to get seeds of tobacco or else anyone can provide some tobacco seeds?
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You try on this link: http://www.tobaccoseed.co.uk/
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The current growth medium I use contains both NAA and 2'4'DA to prevent differentiation completely. What hormones are required to induce rooting? And what is the exact concentration?
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I'm sorry this is not my area of research.
Best regards
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I read some  research article related to radioactive effects on using tobacco products in relation to.... I have some doubt kindly clear the following
1.why the 210Pb shows higher activity compare to 210Po in tobacco (cigarette, bidi brands)?
2. why cigarette ash content register more polonium 210 and lead 210 content ?
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thank you for your kind suggestion and reply your reply is very helpful for my research work
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Which reference genes are appropriate for qPCR quantitation in plant such as tobacco? 
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needs to be another miRNA of course
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i am trying to isolate microorganisms from tobacco callus but i didn't find any microbial growth in nutrient agar,PDA with and without antibiotic.
are calluses free from microorganisms or there are some different media used for isolation of microorganisms from calluses. 
can anybody have an idea about it.
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Thank you Indrayanto for your suggestion  
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I am in the middle of a research about analysis of cigarette smoke. And the instrument that i will use needs samples in liquid form.
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yes, it is better
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I have run CAPM,3factor and 4factor regressions but i couldnt find significant results.Have you any idea of another measure?Someone told me to compare my portfolio with the benchmark characteristic that Wermers used to evaluate the mutual fund performance but it looks difficult to match my portfolio characteristics with these of the benchmark..
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Dear Charis
There are some methods to build a benchmark
1. If your portfolio is whole tobacco industry which includes all the share of tobacco firms in the market whith the same share as the tobacco market shares => you already have the tobacco benchmark, thus you may compare to the other industry or the market composite.
2. If you portfolio includes just some tobacco shares or all tobacco shares but their weights are not same with the industry => you might collect all the data of tobacco shares and calculate the benchmark as the index of all tobacco shares in the same as they are on the market
Until now, the problem is the way you measure an index, there are many methods such as equally weight, or return weight...that you have to define how your portfolio is constructed and adjusted. If you buy all shares in your portfolio at the same weight than you do nothing, thus you have to measure index as the value weight. But, if you implement your portfolio at an equally weight, then after day and day when the market price change, you adjust your portfolio again to the equal => you constrcut the benchmark as the equally weight...
There are some short  ideas i can give you. I honor suggest you find more infomation about construct an index.
Cheers
Canh
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I need a  Arabidopsis/Tobacco Y2H cDNA library to test against two bait protein(Virus proteins). I prepared cDNA library but with it in a Y2H assay there was no interaction seen  against my bait proteins. Now I need the tobacco or arabidopsis cDNA library to check whether any interaction would happen with the bait protein. 
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Hi Sumesh,
You are welcome. 
Yes, I made tobacco root cDNA library by myself. It seemed work well. The protocol I used for making library is from Clontech with # of PT4085-1 (071214) . I find the RNA and cDNA quality is important. The organ and time you collect tobacco/Arabidopsis tissue also matters because your target gene may specifically expressed in certain time and certain organ. The other thing I can think of is the selection stringency, some protein-protein interaction are weak and transient, it is better to try from the lowest selection stringency to higher selection stringency. 
All the best. 
Hongxia 
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I need to know the concentration of nicotine present in my tobacco leaf powder.
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Thank you so much sir for your timely help. 
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We would like to use Bel-W3 cultivar in a course work (ozone-exposure experiment) with MSc students.
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Hi, we would also like to get some Tabaco Bel W3 seeds and protocols for our MSc program laboratory exercises. Did you have any luck finding this historically significant variety?
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I have just received fresh samples of tobacco leaves with white mold on them. So I am trying to ascertain the genetic diversity of these "fungi"
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Preparation and maintenance of pure culture by spore suspension or mycelium fragments  is of prime importance. then you can use mycelium form this culture for DNA extraction.
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My work is on genetic characterization of S. gesnerioides in tobacco and I intend to use SSR markers for characterization studies. I need to check if there could be genetic differences between S. generioides collected from different locations within the same area and across regions since we are getting fresh reports of infestation in tobacco fields. I also need to collect S. gesnerioides from cowpea fields in areas where this strain was reportedly found and compare with the tobacco Striga
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Many thanks for the articles with all the necessary guidelines, I will follow closely what is required. 
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I want to transform tobacco by agro bacterium (GV strain), but my problem is that selection plates only contaminate. I used 500 mg/l cefotaxime in plate 250mg for washing but it can't remove it, and after some days the bacteria covers the explants and kills them. If anybody has any experience that can help me please let me know
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1. Once you see the 'overgrowth' of Agrobacterium on explants (leaf discs), chances are there is no way to turn back. I wouldn't put too much effort trying to remove them. Start another batch.
2. GV strain, such as GV3101, is more virulent than other strains, such as LBA4404. When you use GV strain, you need to keep an eye on 'overgrowth' issues.
3. Transformation:  When I used GV3101 for tobacco leaf-disc transformation (I suppose that you use 'leaf-disc' method), I diluted the Agrobacterium culture 1/10x (diluted with sterilized MS liquid medium) before I dipped the explants into the solution. After dipping, blot them dry.
4. Selection: Two antibiotics were used for selection. For example, if 250 mg cefotaxime needed, I would use 125 mg cefotaxime + 125 mg carbenicillin. I found that such treatment has a better result compared to use only one antibiotic (although no formal study to confirm it has carried out).
5. Selection: Explants on the solid selection medium will become 'curvy' and prevent the explants completely contact with the surface of the selection medium. When you see this occurs, push those explants into contacting the surface of the medium. This needs to be taken care of especially at the beginning of selection because the potency of antibiotics will decrease later, at room temperature.
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is there any standard method for determining COD for solids?
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Dear Randa M. Osman 
Determination COD and BOD for solid material require some operation:
1. firstly you can weigh an amount of solid materials (about 10 - 20 g) .
2. Then you can suspend that  in a distilled water (about 1000 ml).
3. Then you have to mix this suspended solution by a magnetic stirrer (about 20 min).
4. After a settling time (about 1 h) in an Imhoff cone  (1 L), you can take a sample of supernatant for BOD5 and COD as 'standard methods for the examination of water and wastewater' a reference book the same as water and wastewater (may be need to have a centrifuge the supernatant for soluble BOD and COD.
5.Then, you have to convert the result as mg/kg solid material. For example if you put 10 g solid waste for 1 liter distilled water and obtained 100 ppm COD, you can convert it [(100mg×1000g/kg)/10g]=10000mg/kg or 10gCOD/kg solid material.
I hope, the mentioned materials could help you.
Best Regards
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It is clear that the experience of addiction to tobacco is different from the experience of addiction to alcohol, or something like cocaine. If we agree that addiction is socially constructed while also manifesting a physical or (dare I say) psychological dependency, then in what specific ways are commonly associated addictions (alcohol et. al.) really differentiated from tobacco. Intoxication of an altering of subjective experience may be the key to delineating one from the other. Thoughts?
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Physical or pharmacological dependence can probably be explained by the increase, among smokers, of the number of "nicotinic receptors" on the surface of nerve cells. These receptors are called nicotinic because nicotine binds to them very strongly in laboratory conditions (and when you smoke!).But under natural conditions, there is no nicotine in the human body: the nicotinic receptors are in fact designed to receive acetylcholine, which is one of the most common neurotransmitters in the human body, especially within the brain, but also in terms of muscle activation.
So taking nicotine is likely to have effects at conscious and unconscious levels in human beings. Among dependent subjects, studies show that the number of nicotinic receptors decreases slowly after smoking cessation. A normal level is only reached after 6 to 12 months at the earliest.However, the acute effects associated with the physical addiction to nicotine (withdrawal symptoms), disappear 1-2 months after quitting smoking, according to the degree of dependence. For this reason it is important to follow the treatment (medical consultation, nicotine replacement therapy, bupropion) for at least 2 months.
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Many smokers are using waterpipes worldwide, some of them to quit smoking. Smokers seem to perceive waterpipes as less harmful than cigarette smoking and think they will be able to quit waterpipes more easily than cigarettes. What's your experience/opinion about this?
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Dear Virginia
The answer to your question is "No". But I must admit there are no studies that have investigated your question because it is somewhat counterintuitive. Waterpipes combined a boring process of charcoal with the heating of tobacco with a number of additives and the result is an inhaled mixture that contains many more toxic substances than smoke from tobacco cigarettes. Both include nicotine, the main addictive substance in tobacco addiction. 
Many people think that water pipe smoking is less harmful because the smoke is "filtered" through water. The main effect of the water is the cooling and humidification of the inhaled smoke. The other aspect which may suggest it may be a method to quit is that it is used less frequently and mostly in a social context. But real nicotine addicts will hardly be able to reduce nicotine consumption by purely changing to conventional water pipe use.
Regards
Macé
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The article reports the results of comprehensive analysis of dynamics in changes of chemical properties of Cavendish tobacco and organoleptic properties of its smoke during the cyclic process of additional thermal fermentation.
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Hi there,
Try Bill King at the Cancer Council Victoria, in Australia: Bill.King@cancervic.org.au
Regards
David
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I'm looking for database on smokers, not smokers, former smokers by gender, age and type of disease, period 2012 - 2014 for the following countries: France, Switzerland, Austria and Slovenia. Preferably, the samples should have high number: more than 10,000 units.
Someone knows websites from which to download these databases?
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In Researchgate look at Catherine Hill great French specialist on the question: 
- Le tabac en France MAJ tableau 1 à 3 et figure 1 à 8.
- Pubmed:
Ribassin-Majed L, Hill C. Trends in tobacco-attribuable mortality in France. Eur J Public Health 2015; 25 (5):824-8.
Hill C. Trends in Tobacco smoking and conséquences on health in France. Prev Med 1998; 27(4): 514-9.
Hill C. Tobacco Epidemiology. Rev Prat 2012; 62(3): 327-9.
In Researchgate you'll find informations at François Beck other great French specialist in Public Heath and Tobacco consumption and diseases connected with smoking.
Best regards.
Jean.
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I'm looking for database on smokers, not smokers, former smokers by gender, age and type of disease, period 2012 - 2014 for the following countries: France, Switzerland, Austria and Slovenia.
Preferably, the samples should have high number: more than 10,000 units.
Someone knows websites from which to download these databases?
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Hi
Register in the google window:  baromêtre sante tabac en France
Y'll obtain survey of tobacco consumption every 5 years 2005/2010/2015
INPES website:  www.tabac-info-service.fr
Best regards.
Jean.
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I am trying to estimate the proline level of tobacco seedlings under normal and NaCl treatment in Wt and transgenic tobacco lines. In normal conditions the level of proline in tobacco seedling was estimated by using Bates protocol and after calculating the value with the formula given in bates protocol I am getting the lowest value of 23 ug/mg fresh weight. But when I was checking some article. I found that the value of proline is ranging from 1 to 5 ug/mg fresh weight. 
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You might like to see simplest and eco-friendly protocol for proline quantification that appeared online in Protoplasma (you may download the same from my ResearchGate site).
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The results of TCA (Tobacco Cessation Activity) are generally limited to 20 40% achievement in quitting Tobacco. What other regimes / methods can be incorporated / utilized to get more better results?  
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Daily brief vigorous physical exercise must be included.  Even 10 to 15 minutes daily will go a long way to help quit smoking by adlescents and young adults
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I am searching for an easy experiment that can be done in school for detecting certain toxins in cigarette smokes. In order to visualize the experiment as good as possible I would like to avoid premade detection tubes to visualize best how the test is done. I was thinking about the reduction of I2O5 to I2 by carbon monoxide, but I am not sure if there will be enough of an effect to see any result if you just prepare a test yourself. Of course verifying the presence of nicotine or PAH would also be nice. Does anybody have a few key words for me to point me in a good direction?
Cheers,
Christoph
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I was thinking about an experiment with tobacco smoke, to be simple and visual for a class of sudents or pupils. I think the best is to light a cigarette and puss the smoke through a strong light source so that everybody can see the tiny smoke particles, filling the air of the room, which when breathed go into the lungs and  because  they are of the PM2.5 size are trapped in the pulmonary alveoli. Also,  hold a paper filter  (white , coffe filter or chemistry filters) in front of smoke , 6-7 smoke phases are trapped on the paper and give it a black colouration from tar. Collect tar in a vial to show it how black it is, . Then you can say that this smoke particles contain PAH, carcinogenic chemicals, nitro-PAH,  VOCs, benzene, CO, etc. Also, they have polymeric black colour carbonated particulates that trap stable free radicals producing continuously hydroxyl radical which can penetrate into cells and can cause DNA damage. or oxidize lipids in cell membranes. These pictures can be found in books, and projectd on backboard. See my papers on tobacco smoke. in Researchgate. Also, passive smoking can be explained with a smoking a cigarette and allowing to produce secondary smoke which is more dangerous because of lower temperature and pyrolytic burning  in the tip (the smoke covers the cigarette tip prohibiting oxygen to do the oxidizing burning, and in this way is producing more carcinogens. 
Valavanidis A, Professor
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