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What is the best way to find the annealing temperature for the second step of PCR?
I have primer set that has annealing temp. 60-61degree centigrade and GC content 55-60%. First step always gives good band the second step of touch down PCR gives non consistent bands under same conditions. I am trying this since last six months. what should I change ?
Ok, rereading your question I am now thoroughly confused. If your initial PCR is giving you the proper band, what is the purpose of the second touchdown step? If the first step is NOT giving you the proper band, then I suggest skipping it and going straight to touchdown. After all, touchdown is PCR as well, and will amplify (with optimization) anything regular PCR will amplify. Please correct me if I am misunderstanding the situation.Following
What is the relationship of seismic horizontal and vertical component?
Can we use some formula or method to obtain the horizontal component from Vertical?
Dear colleague, I think that it is a very complex problem because it depends on the wave-type content of the seismic signal. This content varies depending on the source the path (attenuation) and the site effects.Following
Can electrons be transferred from FTO to TiO2?
In DSSC an similar applications electrons are transferred from TiO2 to FTO for example, how are electrons transfered from FTO to TiO2 instead? What conditions are needed for this to happen? For example when performing electrochemistry on an FTO/TiO2 electrode, how do electrons reach the TiO2 surface?
A common example is that you have the FTO working as a current collector, the "electrode" support, so-to-speak , and on top of it the "photo electrode" of TiO2, and the TiO2 may be facing an aqueous electrolyte. When you shine UV light on the TiO2, electron-hole pairs = excitons are created, and the holes drive to the water where they oxidise the water molecules, whereas the electrons is driving in the other direction to the FTO.
I think the energy bands of the FTO and the TiO2 must not be too much apart, otherwise the electron cannot pass. You have here a heterostructure multilayer.
This is a general problem because the stoichiometry of the TiO2-delta and FTO-delta determine the position of the energy bands. and it is not trivial to meet the required stoichiometry in metal oxide. Here one has to tune the processing conditions, like sintering in oxidising or reducing atmosphere so as to engineer the necessary position.
It may be even necessary to put a third layer in-between FTO and TiO2 in order to promote the electron = charge transfer from TiO2 to FTO.Following
How to simulate Automatic Generation Control/ droop control for a generator using simpower system in matlab ?
i want to build Automatic Generation Control/ droop control for a generator using simpower system , bt cant really get the idea where should i put the agc / droop controller in generator ?Following
What is the appropriate time in the day to researcher creative?
Researchers working at different times. Some of them during working hours, or at home or laboratories. the work may be in the morning or evening. In your opinions, which is best time for creativity?
Question for discussion, for the purpose of conducting a questionnaire.Following
Can you help me find a resource for ecological functions supplied by tropical plant species?
Our plant database is used for plant system design. We need all plants defined by their ecological services and functions. Do you know of a text or research depicting these characteristics? Thanks, Dan
I'm no expert on this by any means, but I came across the attached paper and it appears to have (or have used) information about ecological functions of a number of tropical tree species.Following
MicroRNA binding sites at mRNA?
Many papers show that microRNA regulates gene expression at the post transcriptional level (If microRNA increases and its target gene decreases or else microRNA decreases and its target gene increase).
My question is whether microRNA and mRNA are both positive regulation. (If microRNA increase and its target gene increase or microRNA decrease and its target gene decrease).
@Ulich - Effects of multiple up-regulated microRNAs on targets is buried in
Transcriptional override: a regulatory network model of indirect responses to modulations in microRNA expression
Specifically Figure 3AFollowing
I would like to extract plasmid from glycerol stock bacteria. After done with extraction i'm unable to find any band on gel. May I know the reason?
Molecular biology, cell culture, genetic, microbiology.
One solution is to take a brute force approach: grow larger / several tubes of bacteria, isolate the plasmid from each, pool the resulting solutions and repellet the plasmid or concentration it by speed-vac (or whatever other method you have available).
You may also want to verify that the correct plasmid is indeed in that strain before doing all this extra work. Perhaps you should perform colony PCR with some plasmid-specific primers. Depending on the primers chosen, this may also address the integration possibility.Following
The informal economy
in countries called subdesarrollados, south america, the informal economy is carrying the weigh of the family economy, this form of survival is a matter of public policy, law or human right that are protecting this population?Following
Do you think that methodological choice matters in urban sustainability assessment?
For many academics, methodological choice are highly influencing the outcomes of urban sustainability assessment and cities benchmarking. If so, how do you explain the fact that there are some cities that consistently stand out and outperform the others no matter how their sustainability is measured? As this is one of my current research investigation, I am really curious to hear from other academics that are addressing this issue and perhaps exchange ideas and experiences.
Just wondering why nobody mentioned http://www.starcommunities.org/ - this is very comprehensive and aims at comparing sustainability between cities. At least it's relevant for someone from Montreal since the city is also trying to use this methodology,Following
Does someone have a comprehensive diagnostic key for monogeneans?
especially marine fish Monogeneans
I would be pleased If someone inform me about that.
I am grateful to you all for the participation and guidance. Your comments were consequent and I found what I wanted.Following
Is there any kinetic model relating bioremediation rate to temperature, pH, Depth & Nutrient?
Good day all.Is there an established single kinetic model relating bioremediation rate to temperature, pH, Depth & Nutrient? If not how can I model one?
glad to hear your detailed responseFollowing
Will the intelligence of a biological computer be artificial, or organic intelligence?
On the 23rd of June 2012 Alan Turing would have been one hundred years old.
His 1950 paper in Mind “Computing Machinery and Intelligence” is one of the most cited where the "father of Computer Science" proposed to change the question "Do machines think?" to "Can machines do what we (as thinking entities) can do?". The core elements of such machines are still electronic processors.
Knowing that “scientists are one step closer to making a biological computer after building basic components for digital devices out of bacteria and DNA” ( http://www.dailymail.co.uk/sciencetech/article-2050500/Biological-computers-soon-reality-scientists-build-basic-components-bacteria-DNA.html ), will “artificial intelligence” retain value as a descriptive name if, and when, biological computers become a technological reality?
thanks for your clear statements.Following
How so I collect of primary data for participatory development and natural resource management?
Guide me in the designing of methodology for the collection of primary data form the rural people regarding NRM and its different indicators through the new approach of participatory development.
If you truly want to be participatory, then you need the insight of the people from the beginning. Tell them your objective and discuss with those willing to help about how to conduct your diagnostic. They will tell you who to ask questions to and how to collect the data you need.
In the attached book, you'll find a great insight of how participatory methods work and 80 different techniques you can use!
You'll see that participatory techniques are not so new since they started developing in the 70's. Paulo Freire is considered one of its pioneer.
What constitutes good quality educational research in evaluating the impact of education for sustainability in higher education?
My colleagues and I just finished a two year study to investigate the influence of education for sustainability across different disciplines and higher education institutions on tertiary students' views, attitudes, knowledge and behaviour towards sustainability and the natural environment. Can anyone please advise what constitutes "good quality" scholarly or academic research in this area??Following
Does 260/230 ratio matter when it comes to sending samples for sequencing?
I am trying to extract RNA from a non-model organism (Malvaceae) for transcriptome sequencing. I have used Nucleospin Plant RNA kit (MN) together with Fruit-mate (TaKaRa), because of probable high contents of polyphenols and polysaccharides.
However, I still have problem getting the 260/230 ratio up to >1,9 using these protocols. Have anyone used this?
- Adding the homogenized sample to the RA1 or RAP turns everything to glue, that I either have to cut to release from filter-tip.
- Using fruit-mate turns the material black and the precipitate is still kind of thick. How does your samples react?
I suppose this might suggest contamination with remaining polyphenols/polysaccharides. Have you tried an RNA clean up step using something like RNAClean XP beads (Agencourt)?Following
If anyone has some good papers with the key words "Judith Butler, media and feminism"would you please share them with me?
I need them for my Ph.D assignment.
I expect that you have read the papers by Judith Butler, but ResearchGate member Angela McRobbie has written about extensively about feminism; in the paper below, she has not specified any keywords but has included the topics you mentioned:
McRobbie, A. (2004). Post‐feminism and popular culture. Feminist media studies, 4(3), 255-264.
But there may be other papers that she has written that may be relevant.
Good luck with your PhD
When stop considering micelles and start considering microemulsions?
At what percentage of ”internal phase” (either water or oil, depending which is the phase added progressively to the system) can we stop considering the existence of micelles and start considering the formation of true droplets? For example, adding 2.5% water to a [Surfactant mixture+Oil] system generates a clear, transparent, single phase. Can this be a microemulsion? Is there such a threshold that differentiates between micelles and microemulsions? Do you know any paper on this topic? What is your opinion on this subject? Thank you.
due to two phases have an interface, micelles should not be considered as a separate phase. There are no interfaces between micelles and the solvent in the sense as follows. When we consider them as interface without inner phase, term interface would lose its sense. At the interface molecules of the phases have asymmetric interactions with molecules of the same phase, i.e. they have also neighbours of the other phase. From this derives interfacial tension, i.e. it is unfavourable to increase the number of molecules in the interface. Further, as far as I understand liquids with highly swollen micelles are also called microemulsions.Following
How far X-ray can be used in therapeutic application for treating cancer in Brachytherapy irrespective of using radio isotopes?
I am intended to know about using x-rays for treating cancerous cells.
Thanks in advance.
It is a matter of interaction of radiation with matter. In brachytherapy beta or alpha emitters are used because they deliver much more energy to matter into a very small path (very high LET). This particles travel very small path into the body (this is the reason they are placed next to the tumor) and deliver very high dose. Using photons (lower LET) instead of heavier particles will deliver smaller dose and hence less effective treatment.Following
Phase noise modelling for 73GHz band?
I am trying to simulate Phase noise model for 73Ghz. I was wondering there is any model that captures the effects of phase noise at such high frequency. Moreover, phase noise power at respective phase noise frequency offsets for 73GHz are available? I havent found any in literature.
Why could I not see protein overexpression even if the mRNA increases?
There's an overexpression experiment that is driving me mad.
I'm trying to overexpress a protein in HCT116. I've cloned the cDNA and tagged it with a Flag sequence to detect it (the antibody for the protein is awful, so I want to use antiflag to be sure). The whole construct has been sequenced and there are not stop codons or mutations. I've checked that the codon usage and it's correct also.
In 293T transfected transciently, the construct is expressed and detected with the antiFlag antibody by western blot nicely. In HCT116 I couldn't dectect the protein neither with stable or non stable transfection (in all the cases, I've made a control of GFP and the efficiency of transfection was about the 70% or more).
Moreover, by qPCR, I can see that the expression of the construct increases 10 folds in my cells transfected but in despite of this, I could not detect the protein with the antiflag (I always put controls for the antiflag antibody with and old construction that I have and it's not a problem of the antibody but my sample). I use 40ug of protein for western blot, so I don't think that is a problem of the protein amount.
I even change the plasmid of overexpression from pMSCV to pBABE with the same results. Any suggestions? Thank you in advance!
I haven't worked with human cell lines, but I have experienced in E. coli that overexpression of proteins will lead to the formation of inclusion bodies, which if not fully denatured (and this may be very hard) can then be removed from sample with the insoluble content from the culture during protein extraction. A more strongly denaturing protocol may increase your recovery, though it may be counterproductive, since if you are overexpressing a protein, you probably want it to have an effect in the cell. In inclusion bodies, these proteins are aggregated and are not active. Instead, see if you can produce a line that has only a modest increase in mRNA abundance as inclusion bodies form more rapidly when large amounts of protein are being synthesized. By slowing growth of the culture - with bacteria, we incubate at lower than optimal temperature - you can ensure that there is protein being produced, just not so much that it overaccumulates. You could also see what happens when you try to detect the protein from a diluted sample of your pellet produced during protein extraction with your antibody. Often antibodies have trouble detecting aggregated protein, but you may get lucky that some of the epitopes are either exposed, or some protein has gotten released during the extraction. This will confirm that the protein is indeed being translated, which we cannot be sure of from the information you provided.
Hope that helps!Following
Does anyone know if there is any research paper on student leadership at secondary schools?
I am in search of this topic for a research on classroom management. Previously I studied the effects of teacher leadership on classroom management. Right now I am trying to observe the effects of leadership of students at schools. Any researcher interested in leadership from different countries are welcome to study together in comparison .
Thanks. I will put together a file and send it to you as soon as possible.Following
What is the result of "Test data" in BCI competition III: data set 2, P300 speller paradigm ?
In BCI competition III: data set 2 there is 2 subject i.e. subject A and subject B.
In both case there is train data and test data. In training data set there is target character but in test data there is no target character. So, after classification of test data with which I should compare the result?
If you're searching for more P300 data, we have publish an open database that you could find here.
I hope, it will be useful for you.Following
Which is the best CAM system that provide 5 axis programming?
5-axis programming of CNC machine is one of the most interesting topics for research in the relevant field. However, it is not obvious which CAM software provide better options to develop 5-axis CNC programs.
ESPRIT is a good option.Following
Can open-book tests/examinations address the problem of cheating? How about allowing students to 'Google' answers?
The embedded post from Faculty Focus points out that students may be tempted to cheat in instances where responses to a question can be easily 'Googled'. It is suggested that open-book tests, including challenging application questions that relate directly to the course material, may help overcome this problem.
Some even believe that students should be allowed to 'Google' information during examinations, for instance, because they have to demonstrate digital literacy (an opinion expressed in the post from The Guardian).
Which of these approaches (if any) are acceptable? What would serve as guidelines for good practice if either of these approaches are incorporated in teaching and learning? Would a particular approach be acceptable in different fields or at various levels of study?
In my opinion evaluating students learning demands new ways of acting in nowadays life! If the instrument used to evaluate challenges them and fosters their creativity and critical thinking, students won't cheat! Let them use textbooks and Google the asnwers , but create forms of evaluating very far from the traditional ones , put them into the position of reflecting their answers,and elaborating proper solutions.
I am one of those that believe students should be allowed to 'Google' information during examinations, if the task is conceived in such a way that they have to apply their skills to find out solid information and process it to elaborate an answer .
Have the students be metacognitive and evaluates their own learning process!!Following
I need some help in detecting cleaved caspase 3, does anyone experience with the cleaved caspase western blot protocol?
I have a question to ask Wang Haisheng, as your explanation that you mentioned 3 bands can be detected ,did you mean that except molecular wight of 17,there is aslo 15,18 those are very close molecular wight to 17?Following
How can I get rid of e-waste?
Up to 90 percent of the world's electronic waste, worth nearly US $19 billion, is illegally traded or dumped each year, according to a new report.
By trying to investment the to raw materials repair or reproduction them.Following
I want to lyse mitochondria for measuring compex 1 activity.Is there any suitable protocol which can release the enzyme without damaging its activity?
mitochondria lysis protocolFollowing
Information greatly appreciated for best NGS data (MiSeq) pipeline for analyzing 16S rRNA sequences from gut: in-sample and between-sample diversity?
Data-preprocessing is done until creation of FastQ files. I am not bioinformatiotion by education, but deal with Linux (not programming (R or other), unfortunately). Also realize that first step is probably Mothur for OTU picking, but what could be next?
Have you had a look at MG-RAST? You can enter fastq directly and got a whole load of output info. It's a web based program so no need command line.Following