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- 11How do i solve my problem if there is no expression of protein after site directed mutagenesis experiments?
I have done site directed mutagenesis in a 4kb plasmid(pQE31) having gene(500bp) encoding 18kDa protein using Quick change lightning site directed mutagenesis kit from Agilent. Transformed the PCR product into XL gold competent cells. Everything worked fine, after that i sequenced my DNA and confirmed the mutation at 52nd amino acid position. When I transformed the DNA into my expression strain M15 E coli cells there was no expression of the 18kDa protein. my wild type protein was also expressed in the same cells using 0.4mM IPTG. I have tried different concentrations of IPTG and low temperature. Also I confirmed no other insertion of stop codons within the sequence. Also there is no change in the promoter and other sequences. what may be the problem?
As Pavel says, if it doesn't express it is critical so you cant mutate. So try to change to some other residue.Following
- 1What is the most accurate device to measure foot sole skin temperature in human subjects?
I'm questioning about the accuracy of infra-red devices...
There are many studies which deal about the use of the infra-red devices (Infrared Thermal Imaging Technique), including the important studies applied to the diabetic neuropathy. Nowadays, we can observe different studies which using this technique of measure and others importants (i.e, wireless system), however, there are the problems as any other device. You should verify the error level of this application, because it's a situation that we probably will find in almost all types of medical devices. In my opinion, the most problem in studies about termography it's in the interpretation of data and mainly in the correct equation or formulas application, such as the standardization of data when needed. So, the most accurate device it will depends of your application, group, objectives, error levels, applicability and reproduction of the data. In any way, I found some papers which I believe it can to clarify your ideas.
- 3Which are the most recent updated conversion factors to estimate effective dose from Computed Tomography?
The conversion factors i am aware of is which is updated by european guidelines 2004, also used by ICRP 103 , where the conversion factor for CT brain is 0.0021. Has there been any update of conversion factors recently?
- 3Is cloning a 9kb 3'UTR for luciferase assay necessary or cloning the regions of 3'UTR-miRNA interaction is sufficient?
Hi, the gene of my interest has a 3'UTR of nearly 9.8kb. I am planing to perform luciferase assay for studying the miRNA-target interactions. So is it okay if i could clone those regions where miRNA is binding to 3'UTR (acc. to the insilico tools) or should I use any other stratergy for the same. Please suggest if I could study the miRNA-3'UTR interaction through any other assay.Hi Dear
During my Master's, we just clone the predicted 3UTR sites for luceferase assay.Following
- 2How can we compare between the Hawks and Doves in the US policy doctrine?
Actually, I started writing a research about the US policy, therefore, I need more understanding and details of this concept.
Use evolutionary game theory, typically the formulation by Smith and Price (1973).Following
- NewIs stratified sampling or spatial random sampling able to deal with spatial dependence of sampled units?
I would like to ask the better choice for sampling a finite population on an area (both large enough, such as households in a political unit). I have seen some published papers and books (e.g. "Sampling Spatial Units for Agricultural Surveys") but I'm still aware of spatial dependence problem.
- NewHow do I further troubleshoot my Western Blot?
I have tried western blot for detecting Arcadlin/PCDH8 from hippocampus of mouse. I used 8% of gel and 5% skim milk for blocking. I have checked the 2nd Ab (I used Ms HRP) and the ECL,both are fine. I have tried to check my membrane using N-cadherin and Beta-actin, both were shown well, but not for the Arcadlin/PCDH8.Following
- 27Is there any insight for a curative treatment in the cause of pediatric atopic dermatitis?
Apart from statutory moisturizers and palliative care leading to corticosteroidal suppresion or calcineurin- or Janus kinase targeted immunosupression, is there any hope on other kinase targets eg. TrkA in paper 25594427 attached, relevant to keratinocytes or skin constituents with possibly less adverse effects? The catch usually is the chronic application setting and a reversible -take it, it stops- stop taking it, it comes back with a vengeance- feature, that would best be minimized.
Is there any value in personalised prick/allegy screen test of an exhaustive antigen repertoire including clothing fibers, food constituents, urban pollutants, less established or suspected allergens?
Taking another twist, eg. paper 26385242 attached, have any "Genetic variants and epigenetic alteration as tools for the molecular taxonomy of AD provided the background for personalized management" ?
I specially did not study this question. Because in therapeutic aspect it is unimportant. It is important only that the mycoplasma be cytoparasitic.
I consider that the mycoplasma role in suppression of immune system at an atopy is minor. But it, apparently, the most frequent provoker of atopic attack.
Though not "private" mycoplasmas provoke an attack, but an another, which have similar (but not identical) antigenes.
If the patient reacts "to everything", it's possible that he is infected by M.hominis or similar. Because ubiquitous mycoplasmas from environment often are similar to it.
If its aggravations happen seldom, but is correlated with a menstrual cycle or discharges of households, it's probable that a problem in U.urealyticum. But I should repeat: for treatment it has almost no meaning.
In a suppression of Th1 immune component of primary importance unconditionally is Chlamydia.Following
- NewTesting coefficients between separate logistic regressions as opposed to using a multinomial logistic regression?
I have a case-control design where logistic regressions are used to examine whether risk factors of a certain type of cancer (cancer X). We decided to examine the associations of certain medication use with cancer X by different histological subgroups A and B. Fitting a multinomial logit (which simultaneously investigates risk of A vs Control and B vs control) and comparing coefficients is the solution I have mostly encountered in literature. However, the way we are presenting the Odds Ratios is by conducting separate logistic regressions on A vs Control and B vs control. This approach has the benefit in that the multinomial logit imposes IIA assumption not required by the separate logistic regressions. My interest lies in statistically comparing the coefficients pertaining to medication use between the 2 separate logistic regressions. Although I have not encountered this in literature (maybe I have not looked hard enough), the solution I am contemplating is to use the 'suest' (Seemingly Unrelated Regression) command in STATA, which is able to estimates a covariance matrix of the coefficients between both models as both models are simultaneously fit. The parameters comparing equality of association can then be tested. My question is whether there is a problem with this approach, the lack of documentation on this method makes me nervous.
Many thanks for any advice,
- 4Has anyone had any sucess with a Dichloromethane substitute in a SPE wash step? If so, what did you use?
I'm working on a T3/rT3 SPE extraction using an anion exchange SPE column. I have stumbled across DCM as a wash step in the literature, but if there is a non-halogenated alternative I'm not aware of, I could sure use the ideas. Thanks in advance!
Aurea, I am extracting triiodotyronine (T3) and its isomer reverse triiodothyronine (rT3) from human serum. Currently, I have a quick conditioning step with water & method, sample load, a ammonium acetate buffer wash (10mM pH 9), 100% MeOH, and finally a 2% formic acid in DCM wash before elution.
Thomas, your suggestion with regards to polarity completely rang a bell! I decided to try diisopropyl ether, isopropanol, ethyl acetate, and 2-methyl THF. DIPE and EtOAc show the most promise thus far. Thank you for the direction!Following
- 3How to calculate concentration of fungal extracts?
extraction of endophytes
In my opinion, you can evaporate the extracts to dryness and weigh them precisely. According to the demand of your research, then, diluted it with organic solvent. And you can know the concentration of extracts from fungi.Following
- 5What are the archaeological correlates pertaining to the origin and development of the Sanskrit Language?
What was the form of a pre-Vedic age Sanskrit? Was it spoken in South Asia alone? If a pre-Vedic Sanskrit came from elsewhere, where did it come from?What, if any, archaeology exists to sustain any hypotheses in this regard?
As an ancient historian I would suggest that it is a common condition, with any age of written history and historical source based reconstruction that the fit or the match with archaeological or material evidence becomes a choice between what actually obtains and what we would like to see as obtaining. However, as a teacher of the subject, I would say that it is from here that true or proper inquiry starts! Sorry about this homiletic-materialism.
Sir, I you coming to the BHU for the alumni year, centenary year celebrations. I am at the department of history, faculty of social sciences.
- 11Translation, Philosophy: Can anyone help me formulate a proposition regarding this field given Steiner and Ricoeur's hermeneutics on translation?
I just started in philosophy and I've chosen to work on the field of language and translation for when I reach my thesis and senior research. I've been reading around and compiled some initial data for a draft I'm making. I have Paul Ricoeur's Hermeneutics of translation where he goes on to explore the hermeneutic model of translation in terms of three main paradigms: linguistic, ontological and ethical, George Steiner's Hermeneutic Motion where he claims that there are four parts of the hermeneutic look at translation (trust, aggression, incorporation, and restitution), and Elizabeth Nield's Translation is A Two-Way Street: A Response to Steiner where she argues that Steiner missed some important elements in his theory (First, Steiner fails to discuss the seductiveness of the text in a theory based on a view of translation as interpretation. Second, Steiner fails to give suffecient weight the personal nature of the interpretifve act itself. And, Steiner fails to discuss the moment of encoding - the moment of writing - which for many theorists is the whole translation.)
I keep on looking for the hermeneutic motion's threefold problem that George Steiner is criticizing in his Hermeneutic Motion but I can't seem to find any. I, also, have yet to conclude whether he is talking about Ricoeur's hermeneutic model of translation in terms of three main paradigms or something else. If he is, in fact, talking about Ricoeur's theory, then should I be doing a paper on steiner attacking Ricouer's paper and use Nield's arguments against steiner?
My problem is that I can't decide how to proceed on the field of philosophy and translation. I am still confused on what problem I should be answering and focusing on. I just need some insights and recommendations on how to proceed.
Thank you very much for these texts! I am still reading and researching on the subject, especially on feminism since I am new to this. I will post an update of my work (in progress) within the week. :)
P.S. "Rape in Translation" will be the title of my work.Following
- 1Does the quadruple mass spectrometer (RGA) detect elements and there isotopes ?
- in quadruple mass analyses at a position (m/z) of an element is composed from many peaks, does these peaks refer to isotopes ?
- Ar gas has 3 stable Ar(40) 99%, Ar(38) 0.6% and Ar(36) 0.33%.
The question is: is it possible to detect the Ar(38) and Ar(36) by quadruple mass spectrometer ?
- 3What better combination between fertilizers for farms?
Vermicompost and biological? Or manure?
Thank you Doctor P. Vasudv Naik,
Farm yard manure is heavily populated with Bacillus subtilus gram positive endosporic bacteria which can solubilize insoluble Phosphate in soils and has ability to inhibit many pathogen fungi which attack the root systems of crop plants.
These probiotic effects are not found for synthetic chemical fertilizer.
In addition the ammoniated fertilizer will acidify soils and also work to decompose the Soil Organic Matter critical for water and nutrition as well as probiosis.
Thanks again for your keen insight.Following
- 11Is the reversible work and the maximum work done by a thermodynamic system the same?This concept comes in availability
- NewCan Friction force in a rotational medium also be accounted as a load torque?
Jdw/dt = To - TL - B.(w)
Where J is the inertia, To is the Applied Torque, TL is load torque and B is friction coefficient. w is the instantaneous angular velocity.
Can i combine the TL and B.(w) as the net load torque [Assuming B is variable with time].Following
- 2What is a mild reducing agent that can operate in water or methanol but won't reduce ketones?
I am trying to reduce an organometallic complex that has a tethered diaryl ketone functional group. Unfortunately aqueous or methanolic NaBH4 reduces the ketone functional group as well.
Any other suggestions would be gratefully acknowledged.Hi Saikat
My post was a bit limiting, but thank you, regardless.
To clarify, I am attempting to reduce an Ir(III) complex to Iridium(I) complex.
- NewWhat are some current research topics for neo-tropical avian researchers?
Areas of interest: Costa Rica - Mountainous parks - Primary Forest - Endemics - Macaws - Citizen ScienceFollowing
- 2How to determine copy number of a gene via Absolute Real Time PCR?
I want to check the copy number of a gene in transgenic plant through Absolute Real Time PCR ? Can any body suggest me the perfect way to determine copy number of Inserted gene through ABSOLUTE REAL TIME PCR ?
I agree with much of what Sachin has stated, but, you would preferably graph log of copies vs Cq values for the standard curve. And, if you find that your target insert in the biological specimen extract and a purified template standard DNA for that same target/insert both amplify at the same (or very similar) efficiency, then you can use Xo = EAMP(b-Cq) to calculate the initial number of target copies per qPCReaction:
where Xo = initial number of target copies in the qPCReaction,
and EAMP = Exponential amplification value = EAMP = 10(-1/m)
(e.g if EAMP = 1.92, your target reaction amplification efficiency is 92%)
and b = y-intercept of the plot of log(copies) vs Cq
(e.g., b = the estimated/forecasted Cq value for 1 copy of target)
and Cq (quantification cycle) is the arithmetically averaged observed Cq value of the amplification of target in each your biological sample extracts from your transgenic plant unknowns.
However, should you find that your sample extracts amplify at a different efficiency than your purified standard does, you can use the attached files to reconcile the two slightly different universes.
If a different machine quantification threshold is used for the standard curve than is used for the biological extracts, the 2nd file attached below includes an adjustment to correct for that as well: 10(sample threshold - absolute standard threshold)Following
- 7How do we identfy dendrological features in archaeological charcoals?
I am looking for peer reviewed English language papers on identifying dendrological features such as ring curvature, ring width, reaction wood, hyphae, heartwood etc. I am mainly looking at deciduous Quercus spp. and Pinus spp. although I am interested in papers that cover other taxa found in temperate to semi-arid environments. The aim of my research is to see if it is possible to identify woodland management via patterns in dendrological features.
That will be helpful! It was great to meet you at Anthraco2015 and hopefully our paths will cross again when I make it to Europe! Fingers crossed my QI paper for the conference gets up!
- 5What caused the obvious capacity fading of Li2S cathode?
I have fabricated the Li2S cathode and make a coincell with the Li metal to test its charge discharge function. As the figure shows. The first charging capacity is somewhat good. But then fading very quickly.
Can someone has meet such problem?
What maybe the cause of this phenomenon? May be caused by the unstablility of my cathode?
Thank you very much!
Hi, Claire, Sridhar,
Thank you for yours help.
The 1st cycle exp up to around 3.6V because usually the Li2S cathode shows high overpotential in the 1st charge, one of the solution is to set high cut-off voltage to active it due to the papers. But actually, I think my sample do not need activation... I have done other experiments with the 3.0 V cut off potential and other samples, the fading phenomenon is the same.
My cathode is not nanostructures, It`s sulfur in the micro size carbon pores. I used chemical reactions to change sulfur cathode into Li2S cathode. And after the reaction, the pore structure seemed collapsed and can fall down as powders... I think the shuttle effect and the low conductivity both caused the rapid fading.... I have no idea how to solve this problem...Following
- 13In your opinion what are the hot topics in finance filed?
I want to know some hot topics in finance filed related to banks, financial, industrial and service sector.
Balance of Payment crisis
Foreign Portfolio Investment
New stock Instruments
Innovative services in BankingFollowing
- 5What kind of foraminifera is that?
Greeting agglutinated salt marsh foram's researchers,
I have encountered in southern California salt marsh the following live&dead species in surface samples. Test flattened, ovoid in outline, planispirally enrolled and its wall is very coarsely agglutinated. I thought it might be Ammobaculites labvthnangensis (Loeblich and Tappan, 1988) but I'm not sure. I would appreciate your advice on that- see SEM images. Thanks:)
Have a look at this link for the original description and lectotype designation of Ammobaculites:
Assignment to Haplophragmoides sp. can be made.
- NewCould anyone help in identifying this crab (family or genus)?
This Crab this crab was found in an estuarine ecosystem in Cartagena, Colombian CaribbeanFollowing
- 3How to calculate strain and crystallite size using topas?
I'm analysing the crystallite size of a ball milled powder, and am not sure about my calculation method using topas.
when I do the calculation using topas, if I don't consider the strain factor, only choose a L or G profile for the peak, and look at the lvob value for crystallite size, then the crystallite size is usually aroun 5 nm;
but when I consider the strain factor, tick both G, L, e0, run the fitting, then tick off G, as it usually gives a large error value.
then do the fitting again with L and e0 factor, the crystallite size increases a lot to over 100nm, is there anything wrong in this calculation? as the size of 5nm is more reasonable (I did TEM also to check the size), but strain should be considered for a ball milled sample right?
Thanks Manjeet and Robert for your help!
and Robert, I think the instrumental broadening is already considered when I put in all those instrument-related parameters in to topas? I've attached the scan itself and the parameters I got from topas when strain is not considered.
actually I checked the discussions on how to determine micro structure on the forum, but still not sure about whether I did it correctly, as I got a big size difference when adding in the consideration of strain.Following
- 6Does anybody know of a good alternative to iCODEHOP as a tool for designing degenerate primers?I am struggling to get hold of iCODEHOP as it apears to be no longer available for download. i have had similar problems with HYDEN. I am looking for a reliable computational tool for degenerate primer design.
Dear Russell John Scott Orr,
I tried to use PRIMACLADE and got the result, but I do not know how to pick up a pair of primer. Please check my attachment file and give me some advice in picking up a pair of primer. Thank you so much.Following
- 2Hi ... Phylogenetic dendrogram of hyssop?
unfortunately i cant find any phylogenetic information about Hyssopus officinalis .
this genus is from Mentheae tribe. and i'm going to find close genus with it.
A few more sequences added. Maximum likelihood tree attached in PDF format.Following
- NewWhat are gravitinos?
In supergravity theories combining general relativity and supersymmetry, the gravitino is the gauge fermion supersymmetric partner of the hypothesized graviton. It has been suggested as a candidate for dark matter.The gravitino is the fermion mediating supergravity interactions, just as the photon is mediating electromagnetism, and the graviton is presumably mediating gravitation. Whenever supersymmetry is broken in supergravity theories, it acquires a mass which is determined by the scale at which supersymmetry is broken.Following