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How can I design a standard curve for methylation detection by methylight real time PCR?
I bought a 100% methylated human control DNA(bisulfite converted) 10 ng/μl & unmethylated human control DNA (bisulfite converted) 10 ng/μl (EpiTect PCR Control DNA Set, QIAGEN). I don't know if i should use only the 100% methylated human control DNA in the standard curve or both of them.
In standard curve design, should i use both primers& probes of the reference gene and the gene of interest or only of the reference gene? If i use the reference gene only, how can i get the value of the gene of interest used in the formula:
[(gene/ALU)sample/(gene/ALU) CpGenome universal methylated DNA]*100
Knowing that i use probes labeled by different fluorescence dyes(FAM& VEC) for the reference and target genes respectively.
Sorry,i didn't mention that i'm using methylight for methylation detection.I corrected the question.Following
What is the best way to crystallize a Large protein (urate Oxidase) at very low concentrations (~16 micromolar)?
I am attempting to crystallize a well known protein urate oxidase. The purified protein solution is 16 micromolar. The protein has a pI of 8.46 and a molecular weight of 39.2 kDa. I am hoping to use a hanging drop vapor diffusion method with PEG (8000 or 20000) to precipitate and crystalize the protein, but am open to using any other methods that may be of interest. Planning on buffering with TrisHCl (50-100mM at pH near pI ~8-8.5).
Thanks so much! Any and all help is greatly appreciated. :)Following
Does anyone know of any research into educating existing teachers to incorporate e-Learning into the lecture theater?
I am interested in incorporating the use of a clicker (audience response systems) into our established large group lecture based curriculum in tertiary education. Many of the younger lecturers have been keen to have a go, but most of the older, more established teachers are reluctant or out-right refuse to try. There is plenty of literature to support the benefits of this technology in this setting, but the uptake has still been difficult.Following
What are some of the theoretical frameworks or models that can be used to study population dynamics, environmental change and human health nexus?
.Hi All, I am planning to research on population dynamics, environmental change and human health nexus in developing countries. I have reviewed some models and would like more recommended materials and advise to polish up my proposal. Warm regards.Following
What is the role of EUS in pancreatic cysts?
- should it be used to characterise all cysts are just those greater then a certain diameter?
- what is its application in surveillance?
- should FNAC be routinely undertaken for all cysts?
- Can characterisation be reliably made by cross-sectional imaging hence reserving EUS for more suspicious lesions?
EUS with FNA is the best tool for a positive diagnosis of pancreatic cyst. It can differentiate benign lesions (pseudocysts, serous cystadenomas) from potentially malignant (mucinous cystadenomas, IPMN) or malignant (mucinous cystadenocarcinomas) with a high degree of accuracy based on image characteristics, biochemical fluid analysis and fluid cytology.
When cysts are small, with no mural nodules and sugestive for branched duct IPMN, FNA is not mandatory. The same can be said for a typical "honeycomb" serous cystadenoma, FNA may not be mandatory.
EUS can also be used for surveillance of branched duct small size IPMNs.
As CT scan or MRI have lesser accuracies than EUS and as EUS allows FNA, I would suggest EUS in all cases to establish an accurate diagnosis.Following
How can I implement elliptic curve encryption and decryption in java?
Actually ,I am looking the code for implementing elliptic curve cryptography(encryption and decryption) in java.
Bouncy Castle is the default option for EC in Java. Not that fast, but works.Following
What inference can be deduced from the fact that the pre-reversal enhancement magnitude during June solstice peaked 1 hour after other seasons peaked?
I am carrying out an investigation on plasma drift during the evening time, I observed that the average PRE magnitude for a complete solar cycle during the June solstice (covering May to July) peaked an hour after all other seasons had peaked. What could be responsible?
The PRE magnitude which peaked an hour later in June solstice than for other seasons could have been as a consequence of the decrease in the equatorial zonal wind and conductivity gradient. The decrease causes delay in the zonal drift reversal, and as such the PRE peak during this season is dragged further into additional time.Following
Which pre-treatments could be applied to fibers, to improve methane yields and avoid inhibitions in a Anaerobic Co-digestion process?
In a Co-digestion process of high lipid content residue and lignocellulosic fibers.
The idea is to improve methane yields and avoid inhibitions.
If possible please provide references.
Thank you for your help ;)Following
What are the best online free global daily weather databases?
Many online data bases have been developed over the last decades. But, many of them are country-specific (specially for the US) and most of them are not free. Do you know any network from which one can download freely the daily weather data (specially temperature and precipitation) on a global network or at a considerable number of weather stations across the globe?
Hello Dr. Ababaei
This website allows you to download daily CFSR data (precipitation, wind, relative humidity, and solar) in SWAT file format for a given location and time period.
International current weather and weather conditions for the past 24 hours have been the second link.Following
How can I calculate the yield of the products from a GC chromatogram which is having retention time and peak area only?
Thanks in advance for your replies.
For calculating yield of all products (one by one), you can use this formula:
Peak area of one peak / sum of peak areas * 100Following
How can I make sure that our platelet population is not contaminated by leukocytes?What is the best way to isolate total RNA from platelets?
RNA isolation from platelets for miRNA studies.
1. Collect blood using Acid citrate dextrose as an anticoagulant. The lower pH (somewhere around 6) will contribute to the maintenance of live platelets without the need for chelating all the calcium ions from the sample.
2. When recoverying your PRP, after spinning the tubes, make sure to capture only the upper 2/3 of it, leaving the 1/3 part to the tube. This lower part is so close to the leukocytes white coat, and could add contamination to your platelet-rich solution.
3. Proceeding to further isolation of platelets through albumin gradient and gel filtration is also advised. Tydode buffer or leukocyte lysis buffer should be up to consideration.Following
Are there recent articles on distributed rough set method in data mining?
I need an updated reference on Distributed Rough Classification Modeling Method or Distributed Rough Set method in data mining. So far, I have experimented on Binary Integer Programming (BIP) ported on Distributed Inter Process Communication (DIPC) distributed cluster. My research is more towards porting Rough set algorithm on a distributed cluster machine. Is there any other Distributed Rough Set method that I have missed? Please advise. Thank you.
You can see one trial of using a parallel rough set method with MapReduce at:
How to use a .dll file in matlab 2013b?
I have a .dll file that is the output of previous versions of Matlab (or I dont know what) written in 2003. I found it and I want to use it in Matlab R2013b. what is the easiest way to overcome this? I have little experience in Matlab, so examples or tutorials are gratefully welcomed.
I found loadlibrary(libname,hfile) for Matlab, but there is no .h file. How can I produce one?Following
Does anyone transfected U251, A172 GBM cell lines with lipofectamine 2000? What was your DNA/ lipo ratio?
I have transfected these cell lines several times, but the efficacy of transfections were below 40%. Does anyone know an optimized protocol?Following
Best and easy way to see neuronal degeneration in brain tissue?
I want to see neurodegeneration in my hamsters infected with Prpsc...can anybody guide me how can i easily see either apoptotic markers in brain homogenate or any fixed tissue techniques?
thanks in advance
You may look at presynaptic and postsynaptic markers because neurodegeneration may happen at the synaptic level and causes synaptic loss. For example, SNAP-25 is a presynaptic marker, also PSD-95 can be used as a posrtsynaptic marker. You can analyze these markers either in the brain homogenates using western blot analysis or by immunostaining of brain microsections.
What is the best way to entirely remove palladium on aryl coupling reactions even in ppm level?
Organic Synthesis, Drug Deevelopment, Process Chemistry.Following
Technical question: How can I increase the conductivity of the formed oxide layer (TiO2) on titanium alloys for getting more clear SEM images?
I am working on Ti alloys, and I face a problem to get a clear SEM images for the oxide layer (TiO2) on the surface of the Ti alloys.
We attempt to use Pt. coating for 50s but unfortunately all the topographies of the surface disappeared, ANY HELP PLEASE?
Thank you Dr. Hamouda for your interest, unfortunately, FE-SEM microscope unavailable. So , I think I should work on reducing the time of coating.
I want to take the experience of scientist in solving this problem to save my time and reduce the number of trials.Following
Is Chalmers' so-called "hard problem" in consciousness real?
In his 2014 book "Consciousness and the Brain: Deciphering How the Brain Codes Our Thoughts" Stanislas Dehaene wrote "Chalmers, a philosopher of the University of Arizona, is famous for introducing a distinction between the easy and the hard problems. The easy problem of consciousness, he argues, consists in explaining the many functions of the brain: how do we recognize a face, a word, or a landscape? How do we extract information form the senses and use it to guide our behavior? How do we generate sentences to describe what we feel?
“Although all these questions are associated with consciousness,” Chalmers argues, “they all concern the objective mechanisms of the cognitive system, and consequently, we have every reason to expect that continued work in cognitive psychology and neuroscience will answer them. By contrast the hard problem is the “question of how physical processes in the brain give rise to subjective experience … the way things feel for the subject. When we see for example, we experience visual sensations, such as that of vivid blue. Or think of the ineffable sound of a distant oboe, the agony of an intense pain, the sparkle of happiness or the meditative quality of a moment lost in thought … It is these phenomena that poses the real mystery of the mind”."
Stanislas Dehaene's opinion is "that Chalmers swapped the labels: it is the “easy” problem that is hard, while the “hard” problem just seems hard because it engages ill-defined intuitions. Once our intuition is educated by cognitive neuroscience and computer simulations, Chalmers’ “hard problem” will evaporate".
Personally, I agree with Stanislas Dehaene's opinion.
I realize that my stating the obvious was not necessary. :)
Have a great evening; it's time for me to go home now.
Which cell line can I use to propagate the retrovirus reovirus?
I am planning to use reovirus to lyse cancer cells.
As I know, L-929 cell line has been used for propagating Reovirus. In case of Retrovirus, I suggest Bosc 23 packaging cell line.Following
Any Strong Verifiable Randomness or Random Function?
I'm trying to find a verifiable randomness source. Its output is random values and it can prove to the public that the output is indeed from the randomness source (not made up).
I found papers like "Verifiable Randomness Functions" by Micali et al. and other similar works. However "Weak Verifiable Random Functions" by Brakerski et al. claims that those verifiable random functions are "weak", i.e. they only seem random when input is random but not when input is adversarially constructed.
I wonder what else verifiable randomness is out there?
Thanks in advance
Thanks for your answer, Saeed.
Brakerski's paper, claiming those verifiable random function are weak, was the reason I post this question. Unfortunately they did not propose a "strong" VRF by their own definition, and I did not see such paper either.Following
Is there a way to increase the fidelity of T7 DNA ligase?
In my reaction involving 5 different DNA pieces which differ in their sticky ends, the T7 DNA ligase seems to be ligating non-complementary sticky ends that differ in one base pair.
The only way you can decrease the likelihood of overhangs with one mismatch from annealing is by increasing the temperature. However, the better move would be to change restriction sites so that the overhangs are more different.Following
Preparation standard curve for lipolytic activity?
I need to prepare standard curve using tributyrin as substrat for lipase activity assay. I don't know I must use how much enzym( mg or g). Can you help me?
thank you so much for your helpFollowing
How can I measure the concentration of sodium acetate, sodium laurate and sodium stearate?
I want to measure the concentration of sodium acetate, sodium laurate and sodium stearate in solution, but they didn't have a characteristic absorption peaks in a UV spectrophotometer.
Read ''Standard method'' book. You can use ion chromatography or use UV spect.Following
How to verify the level of significance?
Please help me choose a correct test to verify my hypothesis. I have a string of daily changes in quotations of US Treasury bonds from 2005 till 2014. I calculated mean and standard deviation. Now i want to verify if a change at one specific day (let's say a drop of 15 b.p.) is significant and at what level.
I guess that with "significance" you mean the statistical significance. This is the probability to observe a more extreme value than a given one.
What you need to get this probability is a probability distribution. This means, you need to know how the daily changes are distributed. This is usually some model with a functional part (in your case this could be a trend, since the changes need not be constant over time) and a random part "e" (that describes the variation of the observed values).
You use the functional part of the model to predict the expected value at the desired day, then you calculate the difference "d" between this expectation and the observed value. Then you can use the probability distribution to find
P(|e|>|d|) = P(e<-|d|) + P(e>+|d|)Following
What recommendation do you have for yellowing of leaves?
My obtained plantlets of tissue culture have yellow leaves. Is there any procedure for this trouble?
Hello Atefe, in my opinion, yellowing in PTC could be due to one or more of the following:
1. Photosynthetic photon flux density (PPFD), high PPFD can lead to photo-inhibition, bleaching, yellowing, stunted growth..etc
2. the quality of the chemicals used.
3. Deficiency of some nutrients such as Fe, Mg
4. Medium PH
5. High sucrose content in the medium (6% or more)
6. long incubation (more than 4 weeks)
7. wrong cultural practices during subculture
8. endogenous bacterial contamination
9. High/low incubation temperature
10. type of gelling agent
Is PCR primer is efficient with few mixed bases at certain positions?
Do we avioid mixed bases in PCR primers?
If you're trying to create degenerate positions in a PCR amplicon, then mixed bases are fine. Ensure that the mixed base positions are at least 10 nucleotides away from the 3' end of the primer to prevent problems in polymerase extension.Following
What are the simplest Dipteran species to be rearing and obtaining much maggots at the same stage?
Could you please tell me how to obtain a lot of maggots at the same stage to achieve a bioassay of a biopesticide against Diptera
So, I need to get a big colony with ability to design a rearing method to separate maggots at the same ageFollowing
Can someone help with unknown protein identification?
I need some help in IP. In my ChIP PCR I am detecting the H3K27me3 enrichment. Now I want to find which protein is responsible for this high enrichment. How could I find out the unknown protein responsible for the increase in the H3k27me3 enrichment in my target genes? ThanksFollowing