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- Hypertetraploid cell line, is possible to do gene editing?
Hi to all! I'm entering the field and so I have a lot of question..
The first one is the model: We are currently working with a cell line that is Hypertetraployd , and I'm wondering if it could be possible to do HDR on this cell lines. As I understand, the system does not have an allelic preference. So, in your opinion could be possible to use this system or I have to switch to another model? Another conceptual question: How the Cas9 recognize preferentially a ssOD instead of the other allele? or it is not? so the frequency of mutation would be quite low?
many thanks for any input....
What is HDR? Homologous recombination?
It depends what you try to achieve, if you just want a modified gene expressed and not a 100% replacement of all copies I see no problem. But if you try for a 100% change I would not use your cell line, the probability of modifying two alleles is already decreased (target probability, probability of repair failure and cell death activation), having a higher ploidy is making it worse.
The guide RNA bound in Cas9 (secondary structure and sequence) recognizes the target DNA of the chromosome based on Watson-Crick pairing. The off target effect number with nickase modified Cas9 is significantly lower than original Cas9 (which cuts as one molecule) so I would recommend that latter.
- Which methods may determine the color of igneous rocks?
In some cases, rhyolite has dark color, like andesite. Why?
Does the effusion process affect the color of rocks?
nice discussion, generally the color of igneous rocks refers to the origin (magma chemical composition) which reflect the element content in the magma, so if the magma is basic (Fe and Mg rich) the rock will be dark color and if the magma felcic (Al, Na, k rich) then the rock color will be lightFollowing
- Causes of high background in sandwich ELISA with no antigen?
I am trying to optimize a sandwich ELISA with a p24 protein standard, and I'm getting very high background (0.4-0.5) in wells that have no antigen. These wells consist of only Capture mAb -> BSA Blocking -> Detector mAb-biotin -> Streptavidin-HRP.
At first we suspected an issue with the capture mAb since we saw some precipitates, but we have recently tested with newly purified capture mAbs and got the same results.
This turns our attention to the biotinylated detector mAb, which was made in March 2012 and labeled in June 2014.
So my question is, what do you guys think are some of the possible causes for the high background in the absence of an antigen? I am trying to gather as many possible directions to this as I can so that I can approach this problem more comprehensively. Thank you in advance for your insight!
Thank you everyone for the insightful responses! Here are some details about the system:
PBS-T (0.05%) manual wash after each stage for 6 times, blot with paper towels.
Blocking is done with 3% BSA (ELISA grade). I have also tried 5% with the same results
Concentrations of the capture mAb, detector mAb, and Streptavidin-HRP have been optimized. For example, Streptavidin-HRP is at 0.1µg/ml.
I will definitely look into the blocking conditions, in terms of type, concentration, pH, etc. There is also the concern of the older detector antibody: Would there be issues with the conjugation since the mAb is now 2 years old?Following
- How can one detect a primary amine in non-aqueous solution?
I'm trying to follow the attached method to detect 1,4-diaminobutane. However, the blank (DMF+TEA+Flurescamine) was giving high peak in HPLC. So, I performed a small test with fluorescence spectrophotometer (Varian Cary Eclipse). Surely the result shows that even only DMF+fluorescamine give a nice peak (excitation-390nm, emission scan - 400-600nm). Please see the attached picture.
I checked three bottles of fluorescamine thinking that they may be contaminated by primary amine but all of them gave similar result. It is highly unlikely that all of them are contaminated. Any thought on this will be appreciated.
Is your DMF amine free? DMF breaks very quickly down to dimethyl amine and formic acid:
(CH3)2NCOH + H2O -->(CH3)2NH + HCOOH
The dimethyl amine will probably react with the fluorescamine giving the positive response.
You can check it with Sanger's test using 1-fluoro-2,4-dinitrobenzene. Mix equal volumes of 1.0 mg/mL FDNB (in 95% ethanol) and DMF and leave it at room temperature for 30 minutes. The absorbance of the reaction mixture is determined at 381 nm and 0.5 mg/mL FDNB in 95% ethanol is used as blank. The blank's absorbance is normally in the region of 0.2 absorbance units, while suitably pure DMF had an absorbance at 38l nm of not more than 0.07 units higher than the blank. You will also be able to use this method to determine you 1.4 diaminobutane, but it may not be as sensitive as the fluorescence method.
You DMF is probably the problem leading to the high background, However you can distill it, but the setup is a bit complex. I can supply you with the details if you need it. I found that so-called fresh DMF still have a substantial amounts of secondary amines in it, that will compromise you assay. I would advise that you use an alternative solvent that does not contain an amine moiety such as DMSO or one of the other two solvents in the article, if possible.
- Does liquor addiction leads to auditory hallucinations?
A common form of auditory hallucination involves hearing one or more talking voices. This may be associated with psychotic disorders.
Dr. Shanmugasundaram P
Phenomenology and the Cognitive Sciences, Volume 9, Issue 2, June 2010, is a special issue on the area of hallucinations; this may help.Following
- Quazi Muhammad Rashed-Nizam asked a question:What are the challanges to work with exotic nuclei? Can anyone please brief me the importanc of exotic nuclei.
Exotic nuclei research.Following
- What specific insights can researchers and experts in GDP, GNI, power distance gather from this chart where SDL is one parameter?
My area of research is learning, but it has some things in common with the research of many other RG members. What specific insights can researchers and experts in GDP, GNI, power distance gather from this chart where SDL (self-directed learning readiness scale, SDLRS) is one parameter? Thanks.
My dear @Miranda, there are no such links available for my country. Google became so hot in searching process, but no linked results as you needed! I will try again, as you know, I do not give up!Following
- How do you cope with fossilized errors and help EFL/ESL students improve their accuracy?
As EFL/ESL teachers, how do you deal with such errors? As you know that fossilized errors are mistakes that language learners know are wrong but keep making.
Dear Benjamin and Sarah, thank you very much for your comments. Making an error file for each student is a good suggestion...Following
- Does the surface roughness have an effect on thermal properties of thin film thermoelectrical materials?
Since sputter deposition belongs to the group of plasma-assisted processes, next to neutral atoms also charged species (like argon ions) hit the surface of the growing film, and this component may exert a large effect. Denoting the fluxes of the arriving ions and atoms by Ji and Ja, it turned out that the magnitude of the Ji/Ja ratio plays a decisive role on the microstructure and morphology obtained in the film
In my opinion, the increase in roughness in general should lead to an increase in the effective length of the sample and hence to increase the heat capacity and the resistance of the film.Following
- Has the philosophical analysis contributed to solve any biological conceptual problems?
Of course the first question would be how many conceptual/empirical problems, of philosophy's interest the biology has? How many of those problems has been solved?
Just in case of any extremist response, what would you say to a biology scientists who thinks that the philosophy cannot solve anything?
THE ONLY WAY TO OBJECTIVELY EVALUATE AYN RAND IS TO READ WHAT SHE WROTE NOT WHAT SOMEONE ELSE WROTE- with one exception--her student Leonard Peikoff presented a thorough summary of her philosophy: Objectivism: The Philosophy of Ayn Rand. Here you can get it all in one place and it is accurate--"partially" examining someone else's alleged summary is very likely to be a waste of time- (I am not saying no one else knows her views but the outsider would have no way of knowing)--For those wanting just epistemology and in more detail than Peikoff's book, her Introduction to Objectivist Epistemology is the place to go--As for Harriman's book, the support of ARI and Peikoff is stated directly in the Preface, page 2. I am disgusted by these ridiculous claims of some type of conspiracy. And I was never a "president" of ARI but I am proud to endorse them with no conflict of interest at all. These ad hominem attacks need to stop. They are obviously mean to divert people from looking at the actual ideas.Following
- Researchers should be philosophers or have a matter of logic. What do you think? In our trip looking for the truth, does researching depend on philosophy or needs logic, and on the other hand, should all facts depends on logic?
I want to mention to Quyang that mathematics is a pure science in the sense it is the most logical amongst all sciences; philosophical definitions appear in cases like concept of zero, infinity etc.
In my own life as a lifelong researcher in biochemistry, the philosophical ideas and speculations appeared in my mind at the end of logical pursuit. I think this is how it generally happens.Following
- Can we use Significant Others in motivating undergraduates for study?
In general, a Significant Other (SO) may be defined as a person who has a significant influence on one’s life. A SO may be a parent, a relative, a friend, a teacher, a superior officer, a coworker, spouse, a lover etc. A SO can motivate a student for study.
Improving motivation for study is essential for increasing student engagement. Only highly motivated students can excel in an Open University (which uses Open and Distance Learning methodology in delivering courses). However, quite often we find substantial number of students with low motivation levels. Often they become dropouts.
Can an Open University use SOs in improving motivation levels of its undergraduates?
Does anybody know of such a programme for SOs implemented in an Open University or a related study?Following
- What is the best method for nonlinear dimensionality reduction?
What is the best method for nonlinear dimensionality reduction?
I just checked your institution. If you are dealing with physical and dimensional variables (as opposed to the social sciences which we had to create measures for the INEFFABLE), then you should do a dimensional analysis and see if you can reduce your variables to non-dimensional factors.Following
- Can anyone suggest me some recommendations for DOE for this situation: factors=7,5 with 2 levels and 2 with 3 levels and 4 interactions defined?
I think that Taguchi its very useful, however, as I do not have much experience using these techniques because my experiments are expensive very time consuming.
Here they are. I made them I-optimal.Following
- Is there any effect of autoclave on 2-4 D?
I am preparing culture medium in lab and used 2-4 D, and placed this culture medium in autoclave, now I want to know if there is any effect of autoclave on Dichlorophenoxyacetic acid (2-4D).
I recommend you look the MSDS of the substance up with the manufacturer for heat sensitivity.Following
- Should academic promotions have a minimum time requirement?
Full-time academic staff are eligible for promotion if they fulfill all eligibility requirements for promotion. Promotion regulations differ from a university to another. What is the time requirement in your university? What is your opinion about the minimum time requirement?
Kamal, you are really lucky. But in our country no rules and one turns grey for turning to professor. actually the weak policies of a weak democracy.Following
- Is there a tool available to remove sequences from a protein alignment based on conserved residues?
I am looking for a tool that I can use to remove sequences from my protein alignments that do not contain highly conserved residues. For example, I would be able to direct this tool to remove any sequence that does not contain an arginine at position 124 in my supplied alignment. Is anyone aware of a tool that can help me with this goal?
Just a bit of additional clarification: I am sequencing an amplicon of a functional gene and generating protein sequence alignments using RDP's fungene pipeline. I want to further screen the sequences by eliminating any that do not contain a selection of conserved residues at various positions.
Thank you all for the replies.
I used biopython to solve my problem. Here is the python program I wrote (it's my first attempt at python so any critiques are welcome):
from Bio import AlignIO
from Bio.Align import MultipleSeqAlignment
alignment = AlignIO.read("MyProt.fasta", "fasta")
goodseqs1 = MultipleSeqAlignment()
goodseqs2 = MultipleSeqAlignment()
badseqs = MultipleSeqAlignment()
for sequence in alignment:
if sequence.seq == "F":
elif sequence.seq == "Y":
for sequence in goodseqs1:
if sequence.seq == "Q":
elif sequence.seq == "R":
elif sequence.seq == "G":
elif sequence.seq == "I":
AlignIO.write(goodseqs2, "SCREENED_SEQS.FASTA", "fasta")
AlignIO.write(badseqs, "Discard.fasta", "fasta")
print("Alignment length %i" % alignment.get_alignment_length())
print("initial # of seqs", len(alignment))
print("seqs passing first screen:", len(goodseqs1))
print("final # of seqs", len(goodseqs2))
print("output files are: SCREENED_SEQS.FASTA and Discard.fasta")Following
- What is the importance of ( df and F ) in one way ANNOVA analysis?
I want to know the how df and F values help in interpretation between group during one way ANNOVA analysis.
finding the difference BETWEEN various gorups is MATTER of ANOVA....
The fact that THOUSANDS of "researchers and professors, and ..." associate the ANOVA with the """"Normal distribution"""" make them for making errors in data analysis
I think the my document in the RG perhaps could be useful: """""ANOVA and Least Squares Estimation Some BASICS!!!! Quality MUST be loved, DISquality MUST be hated""""" (on page 7 I say .....)
ANYBODY must not FORGET that these are survival times!!!!!!!!!!!!!!
SO the problem is still there....Following
- How do you study caste violence in an Indian village?
I want to study violence of castes in an Indian village, with the special focus on the vulnerable community of the village.
Please help me to choose the methods and send some links.
Dr. Michael W. Ross from University of Texas came to our university. He was very kind and knowledgable. He might be able to recommend someone for you to work with or be interested. For, in the U.S. while we don't position it as a class society, if you look at behaviors and laws, it certainly has been for minorities. This can be difficult to address becuase it allows others to deny it is there (e.g. Bill O'Reily).
Also, I went to this woman's presentation at FIU. She was really good and engaged in exploratory research such as yourself. She may have changed universities. "Black Land Loss in the Americas: Toward an Ethnography of Spatialized Justice," by Dr. Keisha-Khan Y. Perry, Brown University.
(P.S. I have found it best to read a couple of their articles before I contact another researcher and 90% of the time I hear back from them)Following
- Is there a body of experts who organize seminar, conferences or workshops on local governance? Specifically on the autonomy of third tier government in the Third World.
Many tanx Michiel on d conferenceFollowing
- Are single-blind reviews better or worse than revealed identity reviews?
I know that there've been similar discussions on RG, but I haven't seen this particular question tackled yet. Suppose you receive a manuscript, where the names of the authors are explicitly provided or otherwise clear. You face the dilemma: either you review it blindly (single-blind) or reveal your identity. What would you go for and why? I tend to follow the fairness principle: either double-blind (most preferably) or revealed identity on either side. Any other ideas? Any arguments for preferring a single-blind review?
I prefer blind review. It's one thing if the reviewer knows the name of the author, sometines it's inevitable. But for reviwers to be as objective as possible it's best not to reveal their identity to authors, neither before the review nor afterwards.
Of course, there are exceptions to the rule when you are the one who asks for the review.Following
- Does anyone have any references on the impact of land-cover/use changes on stream hydrograph?
I'm trying to simulate the effects of landuse/landcover changes on the shape of stream hydrograph using a distributed model. Kindly suggest published papers/articles on the subject. Articles covering the changes in flood potential of a river basin due to changes in land-use/cover or something similar may also be suggested. Review articles/books on this subject, if available, may also be suggested. I hope the answers to this question may accumulate to make a good review on the subject.
Thanks in Advance!!
please share any reference articles / reference books on the topic.Following
- What is the appropriate/standard method to find bulk density of powder mixtures, approximate powder size 20-30 microns?
The MPIF standard method is very useful for powder metallurgy. You can use Standard No. MPIF 04 (free flowing powder ) and and MPIF 28/ (Non- free flow powder) for measurement of apparent density. The Bulk density is very near to apparent density.Following
- Is dental abfraction real, or is it a myth?
For dental abfractions, the theory suggests that the high occlusal loads cause large cervical stress concentrations, resulting in a disruption of the bonds between the hydroxyapatite crystals and the eventual loss of cervical enamel.
Here I don't agree with you. Romeed et al (2012) has shown in study the about the concentration at cervical area in canine teeth and their role in development of abfraction lesions. Therfore, Not necessarily, all canine cervical lesions are abrasive lesions.Following
- Can a human gene be expressed in a mouse cell line under the control of the endogenous human promoter?
I am trying to overexpress 2 human genes under the control of their own endogenous promoters in a mouse cell line without success. The genes can be overexpressed in human cell lines. Do you have any experience or suggestions on that? Thanks!
Let me put it this way: I'm not aware of any human promoter not active in mouse yet but if someone knows of an example I would be very curious to learn which one.
That being said as Feymond mentioned the expression pattern of transcription factors in the mouse might not mimic the expression in your cells in human (plus differences from cell lines and primary cells) and there are regulation layers at the epigenetic level that can be based on long non-coding (lnc) RNAs. And the latter might not exist in mice, might not have the same sequence or work cis and thus only if the gene cassette is integrated into the correct location adjacent to the lncRNA.
So just because your promoter is active per se does not mean it is a good model of your human situation.Following
- Vermi-compost vs. thermo-compost: which one is better as a soil amendment?
It is a common belief that vermi-compost (compost made with earthworms) is better than thermo-compost (microbial decomposition with temperatures as high as 60 0C) in quality and soil amendment properties. I wonder if there is strong scientific evidence to support such a claim.
Nice post by Ricky! I agree, the funtional microbial diversity is a key element besides plant nutrient content.Following
- What epistemologies currently exist for the notions of space and time?
I am designing a study on the nature of space and time from the point of view of relativity and quantum mechanics.
1. There is another thread on this site about causality. As usual there are many points of view but many share my belief that causality should not be given a key role in philosophy of science. The main reason is that it leads to single out a specific phenomenon prior to a change of states. What one actually follows in any experiment is a succession of states, with many parameters changing. Why finger one or another ? Take the desintegration of a meson in several pions (or any other particle), one of the most disturbing phenomon, it does not make real sense to say that the meson is the cause of the pion. What is interesting is what is the cause of the desintegration, at this place and time ?
2. Actually QM has no assumption about time and space. Read any common books, you will find the usual axioms (valid for any measure) and assumptions about the duality matter / fields. QM in his actual interpretation is an empiricist theory : it formalizes (with Hilbert spaces, operators,...) the relations occuring between measures. Space and time have no special places, but for the fact that QM considers successive measures (linked to the change of state). Space is seen only through coordinates. Space and time are commonly presented as linked to observables, thus to operators, but of course this is unconsistent because these operators are not compact : they cannot deliver a finite measure. So the need for a complicated interpretation of reality, with randmness,...
One can have almost all the results (meaning Hilbert spaces, operators, probability,...) deduced rigorously from some assumptions about the way models using quantitative variables are built. So the axioms of QM have no physical content. As it is obvious when it is said that the wave function does not represent a physical phenomenon (except for Bohm).
3. What is of interest in relativity is the concept of proper time. For millenia men have tried to measure time with clocks. So we have a concept of time, linked to observations. But because one cannot compare time as we do for space (one cannot proceed by surveying) time has a physical meaning only for each observer. So if, in spite of that, we want to keep a concept of time one has to 1) define a proper time 2) assume some law to compare proper times. This does not give an ontological meaning to time, it enables only the portability of the measures, and so its efficient use.
4. A thing which is not usually seen is the meaning of physical quantities linked to derivatives with respect to time, such as speed, momentum, force,...As we can measure only the state of a system at a given time, the genuine quantities are those which defines these states, the others (such as speed) are only deduced by comparisons between states. This is where all the machinery of frames appear. An example : contrary to what you read in the books, nobody use an orthogonal frame to locate a point : ask to the pilot of a plane. Frames are used not to locate points, but to measure directions (or angles),. And frames provide vectorial quantities (such as speed). So this a different use of time, meant as the way to compare the rate according to which phenomena changes.Following
- How do we analyse a black-box function?
Black-Box Function Optimization.
Imagine we have a function we are trying to optimize. We do not have the analytical form of the function and can only sample it with a metaheuristic (random search, hill climbing, simulated annealing, genetic algorithms). That is, we treat the function as a black-box. How can you analyse the function, for example
is it “hard/easy” to optimize
how confident can we be in our solution
how much time do we need to spend sampling it to obtain a reasonable solution?
Are there any other ways to look at a function e.g. in terms of landscapes, number of local optima.
Do we have any indication of if we should continue with the current run of our chosen metaheuristic, or would we be better restarting our metaheuristic a number of times and taking the best solution from a number of independent runs. Any ideas/references/suggestions welcome.
The classical engineering approach for characterization of 'black boxes' is impulse response resp. step response.
This primarily applies to "boxes" with 1 input and 1 output, but boxes with more than 1 input can be characterized by scanning the responses of 1 input after the other.
(Boxes with more than 1 output can be considered as more than 1 box.)
Overall analysis and optimization will become anything between 'complicated' and impossible if your black box contains storage elements as they will interact 'unpredictably' (in terms of the 'black' box) with the input signals.
Thus black box optimization must be restricted to relatively simple 'boxes'. Otherwise you will have to 'open the box'. At least partially...Following
- What kind of electrons, free electrons or bounded electrons, occupy energy level in zone theory of quantum mechanics?
In the quantum theory of electrical conductivity are considered free and bound electrons.
the wave function is just a mathematical abstraction-approximation according to one model, moreover QM too is just based on a crude mathematical model since in this way it is easier to get some quantities and this means that it is a bad way to look at the real phenomenaFollowing
- Should a visceral artery aneurysm be treated with endovascular or open surgery?
In your experience what is the best treatment?
I agree with all of you, when possible endovascular approach is a good therapeutic options, even if surgery remain also a good option. What about this article? "Long-term results of surgical treatment of aneurysms of digestive arteries"Following