Vectorization - Science topic

I have created an axis using Datum points with a specific vector in Abaqus, intending to rotate a cylinder around this axis. However, in the Assembly module under Rotate Instance, after selecting the instance I want to rotate, I can only choose start and end points, and the axis option is not selectable. How can I resolve this issue?

we think of a point as a spot in an Euclidean plane

indicating a position.Intuitively we assign arbitrary

framework.The confusion (for me) is when

we consider a line segment.It is made of

infinite points aligned in a certain direction.

So a segment results when all these “ directions”

are added vectorial, but a zero vector is

conceptually an infinite direction entity

so how is this vector direction specified for

a point nathematically. physically of course

we have a visible dot with visible dimension,

Any insight or explanation from all sources

is welcome.

please anyone tell me why this happens

Hello fellow researchers,

I am currently encountering significant challenges in a classical cloning procedure and am in need of your expertise. Here is a brief overview of my process:

Image of Gel After Digestion: Image attached

I am also curious why, despite the removal of the insert during vector digestion, there is no apparent reduction in size on the agarose gel. Additionally, running lower concentrations on a new gel seems to still indicate the original size (6500 bp) and the absence of a double band (second image).

Given this context, I am puzzled by the overgrowth of the negative control and the absence of the insert in PCR screenings. Could there be an overlooked factor in the ligation or transformation steps that might explain the high background of vector religation?

I would greatly appreciate any insights or recommendations on potential adjustments to my protocol that could help in achieving successful incorporation of the insert. Could there be additional checks or modifications to ensure the effectiveness of the phosphatase treatment or the ligation efficiency?

Thank you in advance for your assistance!

Hi im on my final project for my thesis and i get stucked in cloning phase. My goals is to inserting one gene to vector who has another gene too. So i have a gene called llm2-mutan and llm1 who's completely ligated with pet28a(+) as my vector/backbone. I'm trying everything like ligating in 16 C overnight, 22 C 3h, etc. My gene llm2-Mutan was a pcr product. Everytime i checking my colony using PCR with electrophoresis is always showing 1100 bp, but my goals is 2100 bp because i must insert 2 gene in one vector. I hope someone can helping me with my issues. Thank u

hi there . i'm currently working on a thesis which involves designing a fast nonsingular terminal sliding-mode controller for attitude control of a quadrotor. the outputs of sliding mode controller which are three control inputs u2,u3,u4 are used as inputs of attitude dynamic subsystem which in turn generates euler angles for position dynamic subsystem. and prior to sliding mode controller block we have a kinematic inversion block which generates reference euler angles for controller to track. as you may know, on the right side of the euler angles dynamic equations we have a combination of the components p,q,r,omega_r,u2,u3,u4,Jr,etc.

we define the term (omega_r) as follows :

omega_r=w1-w2+w3_w4

in this equation , wi is the angular velocity of i'th rotor . each two rotors which rotate in the same direction (counter-clockwise or clockwise) appear with the same sign in the above equation. and on the other hand we know that vector of squares of angular velocities of rotors is equal to a 4*4 matrix multiplied by a vector consisting of four control inputs. each of four inputs should be designed in such a way that make the left side of the equation( i mean wi^2) greater than or equal to zero, because if it becomes negative, then complex values are achieved for angular velocities of rotors which are completely meaningless. and this is exactly the error that i'm facing with in the simulink :'' Domain error. To compute complex results from real x, use 'sqrt(complex(x))''. and i should mention that i used to achieve reference values for fi,teta,sai by a sin^-1 block but then i wondered that the possible cause for this error might be that the term inside the sin^-1 gets greater than 1 or less than -1 so it outputs complex results for reference values of angles . so instead of designing the kinematic inversion block based on sin^-1 , i decided to design it based on tan^-1 block. but even after doing this, the debugger also keeps giving the same error. so it attributed this error to the fact that maybe u(vector of ui's) is changing in such a way that makes the left side of the aforementioned equation negative. and if we want matlab to calculate square root of left side, instead of sqrt(x), we should write sqrt(complex(x)) which is wrong because complex values for angular velocitis are meaningless in our thesis. i have attached a photo of my own calculations for better understanding.

i would appreciate any suggestion of yours on how to fix this error or where might this error originate from.

Hello,

I am new user of DIANA FEA. I am trying to analyse a masonry structure.

But I am getting this error message:

GEOMETRY: NR=1

SEVERITY: ABORT

ERROR CODE: /DIANA/LB/DS30/2236

ERRORMSG.A: Can't normalize null vector.

DIANA-JOB ABORTED.

Can you help me please?

"How do we understand special relativity?"

The Quantum FFF Model differences: What are the main differences of Q-FFFTheory with the standard model? 1, A Fermion repelling- and producing electric dark matter black hole. 2, An electric dark matter black hole splitting Big Bang with a 12x distant symmetric instant entangled raspberry multiverse result, each with copy Lyman Alpha forests. 3, Fermions are real propeller shaped rigid convertible strings with dual spin and also instant multiverse entanglement ( Charge Parity symmetric) . 4, The vacuum is a dense tetrahedral shaped lattice with dual oscillating massless Higgs particles ( dark energy). 5, All particles have consciousness by their instant entanglement relation between 12 copy universes, however, humans have about 500 m.sec retardation to veto an act. ( Benjamin Libet) It was Abdus Salam who proposed that quarks and leptons should have a sub-quantum level structure, and that they are compound hardrock particles with a specific non-zero sized form. Jean Paul Vigier postulated that quarks and leptons are "pushed around" by an energetic sea of vacuum particles. 6 David Bohm suggested in contrast with The "Copenhagen interpretation", that reality is not created by the eye of the human observer, and second: elementary particles should be "guided by a pilot wave". John Bell argued that the motion of mass related to the surrounding vacuum reference frame, should originate real "Lorentz-transformations", and also real relativistic measurable contraction. Richard Feynman postulated the idea of an all pervading energetic quantum vacuum. He rejected it, because it should originate resistance for every mass in motion, relative to the reference frame of the quantum vacuum. However, I postulate the strange and counter intuitive possibility, that this resistance for mass in motion, can be compensated, if we combine the ideas of Vigier, Bell, Bohm and Salam, and a new dual universal Bohmian "pilot wave", which is interpreted as the EPR correlation (or Big Bang entanglement) between individual elementary anti-mirror particles, living in dual universes.

Reply to this discussion

Fred-Rick Schermer added a reply

Abbas Kashani

A lot to work with, Abbas.

However, I am standing in a completely different position, and want to share my work with you. I hope you are interested about this completely distinct perspective.

My claim is that Einstein established a jump that is not allowed, yet everyone followed along.

Einstein and Newton's starting point is the behavior of matter through space. As such, one should find as answer something about the behavior of matter moving through space, and yet Einstein did not do that.

To make the point understandable quickly, Einstein had not yet heard about the Big Bang yet. So, while he devised his special relativity, he actually had not incorporated the most important behavior of matter through space.

Instead, he ended up hanging all behaviors of matter on spacetime. It does not matter that his calculations are correct.

--

Let me find a simple example to show what is going on.

We are doing research on mice in a cage, and after two years we formulated a correct framework that fully captures all possible behaviors of these mice in the cage. That's the setup.

Now comes the mistake:

The conclusion is that the cage controls the mice in their behaviors.

Correctly, we would have said that the mice are in control of themselves, yet the cage restricts them in their behavior. We would not say that the cage controls the mice.

Totally incorrect of course, and yet that is what Einstein did. He established a reality in which matter no longer explains the behavior of matter through space, but made it space (spacetime) that explains the behavior of matter. It is a black&white position that has to be replaced by the correct framework (which is a surprise because it is not based on one aspect, but on both aspects).

--

I know I am writing you from a perspective not often mentioned, and it may not interest you. I'll find out if you are interested in delving deeper into this or not.

Here is an article in which I delve into this matter more deeply:

Article On a Fully Mechanical Explanation of All Behaviors of Matter...

Wolfgang Konle added a reply

"Richard Feynman postulated the idea of an all pervading energetic quantum vacuum. He rejected it, because it should originate resistance for every mass in motion, relative to the reference frame of the quantum vacuum."

Richard Feynman's idea is perfect, and there is no reason to reject it. The existence of vacuum energy, or better dark energy is consistent with Einstein's field equations with a positive cosmological constant.

The energy gain from mass or energy in motion leads to an increasing dark energy density.

The only idea which is missing, is the answer to the question: What happens with the additionally gained energy density?

As an answer to that question I propose the following working hypothese:

This energy is used to recycle star fuel from black holes.

On a first glance, this answer looks as being pure madness, because black holes with their unconvincible gravity seem to be a deposit of matter for eternity.

But in fact there is a plausible possibility. This has to do with the negative energy density of gravitational fields and the non-existence of a negatively definite energy density.

But we need open minded thinking in order to delve deeper into details.

Sergey Shevchenko added a reply

"How do we understand special relativity?"

- the answer to this question, which is really fundamental one, since is about what is some physical theory as a whole; what really means – why and how the postulates of a theory, in this case of the SR, really are formulated, and why and how the postulates

- which in any theory fundamentally – as that happens in mathematics, where axioms fundamentally cannot be proven – aren’t proven; while are formulated only basing on some experimental data, which fundamentally prove nothing, though one experiment that is outside a theory prediction proves that this theory is either wrong, or at least its application is limited.

Returning to the SR, which is based on really first of all four postulates – the SR-1905/1908 versions relativity principle, SR-1905 also on the postulate that light propagates in 3D XYZ space with constant speed of light independently on light source/ an observer’s speeds; and, additionally,

- in both theories it is postulated (i) that fundamentally there exist no absolute Matter’s spacetime, and (ii) - [so] that all/every inertial reference frames are absolutely completely equivalent and legitimate.

In the standard now in mainstream physics SR-1908 additionally to the SR-1905 it is postulated also that observed contraction of moving bodies’ lengths, and slowing down of moving clocks tick rates, comparing with the length and tick rates when bodies and clocks are at rest in “stationary” frames, is caused by the “fundamental relativistic properties and effects”, i.e. “space contraction”, “time dilation”, etc..

Really from yet the (i) and (ii) postulates any number of really senseless consequences completely directly, rigorously, and unambiguously follow, the simplest one is the Dingle objection to the SR;

- from this, by completely rigorous proof by contradiction completely directly, rigorously, and unambiguously it follows , first of all, that

- Matter’s spacetime is absolute, that so some “absolute” frames that are at rest in the absolute 3DXYZ space can exist, while applications, i.e. measurements of distances and time intervals, of moving in the space inertial frames aren’t completely adequate to the objective reality; and

- there exist no the “relativistic properties and effects”.

Etc. However really the SR first of all is based on the indeed extremely mighty Galileo- Poincaré relativity principle.

That is another thing that

- according to SR-1905 relativity principle there is some extremely potent entity “light”, the constancy of which for/by some mystic reasons/ways forces moving bodies to contract and moving clocks to slow down tick rates; and

- the SR 1908 relativity principle is practically omnipotent, so the moving frames, bodies, clocks for/by some mystic reasons/ways really contract/dilate even evidently fundamental space and time.

All that above in the SR really is/are only postulated illusions of the authors, nonetheless, again, the Galileo- Poincaré relativity principle is really . extremely mighty, and the SR indeed in most cases at everyday physical practice is applied in completely accordance with the objective reality. The fundamental flaws of the SR reveal themselves only on fundamental level.

The post is rather long now, so here

Cheers

Sergey Shevchenko added a reply

So let’s continue about what is “special relativity”

In the SS post above it is pointed that Matter’s spacetime is fundamentally absolute, however to say more it is necessary to clarify - what are “space” and “time”, just because of the authors of the SR – and whole mainstream physics till now - fundamentally didn’t/don/t understand what these fundamental phenomena/notions are, the really mystic and simply fundamentally wrong things in the SR were/are introduced in this theory.

What are these phenomena/notions, and what are all other really fundamental phenomena/notions, first of all in this case “Space”, “Time”, “Energy”, “Information”,

- and “Matter”– and so everything in Matter, i.e. “particles”, “fundamental Nature forces” – and so “fields”, etc., which is/are fundamentally completely transcendent/uncertain/irrational in the mainstream philosophy and sciences, including physics,

- can be, and is, clarified only in framework of the Shevchenko-Tokarevsky’s philosophical 2007 “The Information as Absolute” conception, and more concretely in physics in the SS&VT Planck scale informational physical model, in this case it is enough to read

More see the link above, here now only note, that, as that is rigorously scientifically rationally shown in the model, Matter absolutely for sure is some informational system of informational patterns/systems – particles, fields, stars, etc., which, as that is shown in the model, is based on a simple binary reversible logics.

So everything that exists and happens in Matter is/are some disturbances in the Matter’s ultimate base – the (at least) [4+4+1]4D dense lattice of primary elementary logical structures – (at least) [4+4+1]4D binary reversible fundamental logical elements [FLE], which [lattice] is placed in the Matter’s fundamentally absolute, fundamentally flat, fundamentally continuous, and fundamentally “Cartesian”, (at least) [4+4+1]4D spacetime with metrics (at least) (cτ,X,Y,Z, g,w,e,s,ct); FLE “size” and “FLE binary flip time” are Planck length, lP, and Planck time, tP.

The disturbances are created in the lattice after some the lattice FLE is impacted, with transmission to it, by some non-zero at least 4D space, momentum P[boldmeans 4D vector] in utmost universal Matter’s space with metrics (cτ,X,Y,Z). The impact causes in the lattice sequential FLE-by-FLE flipping, which, since the flipping cannot propagate in the lattice with 4D speed more than the flipping speed c=lP/tP [really at particles creation and motion c√2, more see the link, but that isn’t essential here].

Some FLE flipping above along a direct 4D line can be caused by a practically infinitesimal P impact; but if P isn’t infinitesimal, that causes flipping FLE precession and corresponding propagation of the “FLE-flipping point” in the 4D space above along some 4D helix,

- i.e. causes creation of some close-loop algorithm that cyclically runs on FLE “hardware ” with the helix’s frequency ω, having momentum P=mc above, mis inertial mass, the helix radius is λ=λ/P;

- and the helix’s 4D “ axis” is always directed along P – particles are some “4D gyroscopes”.

The post is rather long already, so now

Sergey Shevchenko added a reply

So let’s continue about what is “special relativity”.

In the SS posts above it is pointed that everything that exists and happens in Matter is/are some disturbances in the Matter’s ultimate base – the (at least) [4+4+1]4D dense lattice of FLEs, which [lattice] is placed in the Matter’s fundamentally absolute, fundamentally flat, fundamentally continuous, and fundamentally “Cartesian”, spacetime,

- and that happens always in utmost universal “kinematical” Matter’s space with metrics (cτ,X,Y,Z), and corresponding spacetime with metrics (cτ,X,Y,Z ct), where ct is the real time dimension.

At that particles, most of which compose real bodies, at every time moment exist as “FLE –flipping point” that move along some4D helixes that have frequencies ω, having 4D momentums P=mc, m are inertial masses, a helix radius is λ=λ/P;

- and the helix’s 4D “ axis” is always directed along P – particles are some “4D gyroscopes”.

So in Matter there exist two main types of particles – “T-particles”, which are created by momentums that are directed along the cτ-axis [more generally – by 4D momentums cτ-components, but here that isn’t too essential], and so, if are at rest in the 3DXYZ space, move only along cτ-axis with the speed of light, and at that a T- particle’s algorithm ticks with maximal “own frequency”, the particle’s momentum is P0=m0c, where, correspondingly, m0 is the “rest mass”.

If a such T-particle, after some 3D space impact with a 3D space momentum p, moves also in 3D space with a velocity V, having 4D momentum P=P0+p, its speed along the cτ-axis decreases by the Pythagoras theorem in (1-V2/c2)1/2 , i.e. in reverse Lorentz factor,

- and, at that, despite that the helix’s frequency increases, the algorithm is “diluted by “blank” 3D space FLEs flips. So the “own frequency above” decreases in Lorentz factor, so the algorithm ticks slower; and so, say, moving clocks that are some algorithms as well, tick slower in Lorentz factor as well; if a particle algorithm has some defect, and so at every its tick it can break with some probability, so the particle is unstable and decay, such moving in 3D space particles live longer.

Nothing, of course, happens with time, there is no any the SR’s “time dilation”.

The post is rather long already, so now

Cheers

Roggers Waibi added a reply

As proposed by Albert Einstein, Special relativity fundamentally transforms our understanding of space, time, and the nature of reality. At its core, special relativity postulates two key principles: the constancy of the speed of light and the relativity of simultaneity. The former states that the speed of light in a vacuum is the same for all observers, regardless of their relative motion. This principle defies common intuition but has been rigorously confirmed by experiments. The latter principle, the relativity of simultaneity, suggests that events that appear simultaneous to one observer may not be simultaneous to another observer in relative motion. Special relativity introduces the concept of spacetime, wherein space and time are intertwined, and observers in relative motion will experience time dilation and length contraction effects. These phenomena have been validated through numerous experiments, such as the famous Michelson-Morley experiment and subsequent tests involving particle accelerators and high-speed particles. Special relativity forms the basis for modern physics, influencing fields ranging from particle physics to cosmology, and challenging our intuitive notions of space and time.

Christian Baumgarten added a reply

Article The Simplest Form of the Lorentz Transformations

Waibi's answer is correct. However, the postulates of SR do not generate understanding. The referenced paper provides evidence that the math underlying SR can mostly be obtained from Hamiltonian notions. Since Hamiltonian concepts are universal in dynamical systems, the mathematical relations of SR are universal as well.

Sergey Shevchenko added a reply

Rather detailed consideration of what the SR is see in series of SS posts in this thread sister https://www.researchgate.net/post/How_do_we_understand_special_relativity/2, pages 1,2;

So here only a few notes to

“…Special relativity introduces the concept of spacetime, wherein space and time are intertwined, and observers in relative motion will experience time dilation and length contraction effects. ..”

- really the concept of spacetime, wherein space and time are intertwined is fundamentally wrong.

Matter’s spacetime is fundamentally unique, fundamentally absolute, fundamentally flat, fundamentally continuous, and fundamentally “Cartesian”, ( [4+4+1]4D spacetime with metrics (at least) (cτ,X,Y,Z, g,w,e,s,ct), where all dimensions fundamentally are independent on each other; utmost universal – “kinematic” spacetime has metrics (cτ,X,Y,Z,ct),

- and, of course, fundamentally there cannot be any intertwining of any dimensions, any “time dilations”, “space contraction”, etc.

So that

“….These phenomena have been validated through numerous experiments, such as the famous Michelson-Morley experiment and subsequent tests involving particle accelerators and high-speed particles… “

- really is quite incorrect. No any “intertwining” , “time dilations”, “space contraction” weren’t experimentally observed – that is fundamentally impossible. All what indeed is observed is/are real contraction of moving bodies lengths, slowing tick rates of moving clocks and intrinsic processes rates in unstable particles,

- but bodies aren’t “space” – though, of course are in space; clocks aren’t “time” though, of course tick in time, which [space and time] compose fundamentally only an empty container where everything in Matter exists and changes.

Though that

“….Special relativity forms the basis for modern physics, influencing fields ranging from particle physics to cosmology, and challenging our intuitive notions of space and time…”

- is essentially correct, since the SR is based on the indeed extremely mighty Galileo-Poincaré relativity principle; and so in everyday physical practice the fact that in the SR the relativity principle is absolutized up to absurd/illusory real interactions of particles, bodies, reference frames, etc., with space/time/spacetime is inessential. Again more see the pointed above SS posts in the linked sister thread.

Branko Mišković added a reply

Please read the file uploaded.

… Read more

The position, motion and acceleration of physical objects in the natural laws understand a relevant reference frame. In this sense, the two extreme solutions have been mutually confronted: an absolute cosmic frame or certain equivalence in a class of the frames at least. The imagined formal frames need be connected to evident material bodies. Owing to the complex mutual motions of all celestial bodies, none of them deserves a privileged status. Not only that the vacuum medium is inaccessible by instruments, but its nature and existence are questionable. On the other hand, even the limited equivalence of the formal frames, in the special relativity, does not obey some exceptional technical situations. A few restrictions of relativity in the mechanical and EM processes are here presented. As the synthesis of the two extreme theses, Mach understood a local orientation, in relation to the dominant material surroundings. In the technical practice, such a frame is connected to Earth. The astronomy and astronautics are tacitly referred to the local surroundings, just determined by nearby celestial bodies or their gravitations.

Aim: The solution of one of the crucial questions in physics.

Study design: Comparison and relation of known physical facts.

Methodology: Exhaustive reexamination of these relations.

Study duration: Throughout the author’s working life.

Results: Instead of the absolute or arbitrary orientation, the local preferential frames are finally affirmed and applied.

Keywords: Absolute, Relative, Local, Frame, Motion

Physical laws describe the object interactions, in the form of the forces or energies determined by the kinematical quantities. The material bodies or particles, as the objects, represent the concentrated amounts of respective substantial quantities. The static, kinetic and dynamic forces are respectively determined by the positions, motions and accelerations of such objects [1,2]. The three mentioned kinematical quantities need be determined in a relevant reference frame, connected to an evident material body. With respect to the perpetual mutual motions, rotations and revolutions of all celestial bodies, the relevant reference is very questionable. Not only that such a frame is due to determine all the forces and energies, but also the object trajectories. Though theoretically simplest, the absolute reference frame has not been identified. At least approximately, the local technical orientation usually concerns Earth. Even after substitution of the terrestrial by solar orientation, it is not adequate in the wider cosmic space, out of the solar planetary system.

With respect to the limited former sights, the initial problem took the cosmological sense. Irrespective of the physical laws, a comparative body played the role of the cosmic center. In the first view, Earth seemed to be such a center. However, the paths of the other planets in this frame are extremely complex and illogical. Instead of the irregular planetary paths in relation to Earth, their concentric, circular or slightly elliptical orbits around Sun, obeying the three Keppler’s rules, were the bases for reliable formulation of the law of gravitation. On the other hand, such Moon’s orbit around Earth appears similarly complex in relation to Sun. Overlooking this fact, the traumatic transition from the narrower into wider references gave the impression of a great scientific revolution. In the final instance, none of these two references is applicable in the wider cosmic space, pointing to the predominant material surroundings, in the given spatial domain or respective level of observation.

A similar problem appeared in electrodynamics. Though the terrestrial orientation satisfies the usual technical practice, the reference of light propagation in the wider space is problematic. In the aim of confirmation of the expected absolute reference, connected to the rigid vacuum medium, Michelson made the known experiment, proposed by Maxwell. The difference of the two relative speeds of light in the longitudinal and transverse directions in relation to the orbiting Earth had been calculated in advance. However, the negative practical result pointed to the local preferential frame, connected to Earth. With respect to the traumatic Copernican U-turn, this fact has not been noticed nor emphasized. The later more accurate results, in amount of a few percent of the calculated value, have not been explained but are tacitly neglected or even forgotten by the time. Instead of the preferential local frames, the arbitraryorientation is proposed and imposed, as the provisory alternative at least.

In the absence of a unique reliable frame, the attention is paid to the mutual relations between two arbitrary frames, which needed equivalence is expressed by the principle of relativity. However, this principle is strictly satisfied by the static forces only, dependent on mutual distance of two interacting objects (Fig. 1), as the difference of their positions in the two frames. The kinetic force in Ampere’s law depends on the speed product [1], inexpressible by the relativity. Including one speed into the field expression, the relativity concerns mutual motion of the magnet and conductor. The dynamic forces, without the other explicit object, are especially problematic. The relativity must be thus restricted to uniform rectilinear motion. The obtained relations of the distances and times pushed back the orientation itself, and the geometrically expressed gravitation did not at all exceed the restrictions of relativity. The two unresolved problems are thus shadowed by the two useless theories.

Restricted Relativity

With implicit idea of relativity, Newton made the wellknown experiment for checking the needed frame equivalence. Rotating a container with water, he observed the radial forces manifest by the concave water surface. Comparing the stationary and transitional kinematical states, he reliably excluded the force dependence on the container rotation, but referred it to the undetermined wider surroundings. In the absence of the precise answer, he assumed a unique and rigid cosmic frame. Not being theoretically determined nor practically confirmed, this frame is the subject for the further reexamination.

Looking for a better solution, E. Mach asked the known double question. Would the water surface shape be influenced by the more massivecontainer, or even by the rotating cosmos? The former part suggested the hierarchy and fractional sum of more local frames, but latter one expected the equivalence of the two extreme frames, already denied by Newton. However, the known fact calls in question the former thesis at least. The forces acting on its satellites do not depend on the rotation of Earth, as the container carrying their orbits around Sun.

A similar result Faraday obtained in electrodynamics. Rotating a conducting disc in the front of cylindrical magnet, he noticed the kinetic induction [1] between the sliding contacts placed in the center and rim of the disc (Fig. 2). However, the magnet rotation was ineffective at all, so that the common rotation gave the same former result. Touching the magnet by the contacts directly, its conducting body took over the role of the disc, with the same induction. Alike the celestial case, this one does not satisfy the relativity. Unlike the former example, not strictly reconsidered, this one can be explained.

Figure 2. Faraday’s kinetic induction

The relativity here concerns the difference of the kinetic and dynamic inductions (1). The former of them is ascribed to the object motion (v) through the present magnetic field (B).

The speed of the causing electricity is implicitly contained in the field expression. On the other hand, the speed (U) of the field itself determines the dynamic induction.

v × B + B × U = (v – U) × B (1)

However, the objects and domains of appearance of the two inductions essentially distinguish. Unlike the kinetic induction affecting the moving electricity only, dynamic one affects all the present electricity. Moreover, the former induction concerns all the object motion perpendicular to the field, but latter one is also conditioned by the field gradient [1]:

∇ × E = U · ∇B = – ∂B/∂t (2)

Figure 3. Kinetic and dynamic inductions

Figure 3 compares the two EM inductions. The current in the central conductor is followed by the circular magnetic field. The transverse motion of a parallel or perpendicular object conductor causes the kinetic induction. Such motion of the carrier, along the field gradient, causes the dynamic induction in the parallel objects only. In the transverse (or circular directions Fig. 2), the field gradient equals to naught. Similar relation may be ascribed to the gravitation on the celestial orbits. The field rotations transverse to their gradients are irrelevant.

Light Propagation

All waves, from the mechanical, via sound up to EM ones, as the medium disturbances, propagate at the speeds determined by the elasticity and inertia of respective media [1]. Unlike the former two waves, concerning matter consisting of the particles, the last wave type also propagates through the vacuum medium. Though not explain this medium, the waves demand its existence. The addition of matter, increasing the medium density, causes the slower propagation. The explanations of various physical forces [2] finally rely on respective media. Though light consists of photons, as the energetic particles, it propagates at the wave speed, determined by Maxwell’s relation (9b). The negation of vacuum medium – by special relativity, does not offer any other explanation of the above phenomena. The propagation of each disturbance is referred to the medium, irrespective of motion of passive observers or their instruments.

The same speed of light relative to the moving devices, or to their formal frames and passive observers, cannot be understood nor explained. Michelson’s result, just being referred to Earth, can be generalized to the other celestial bodies, but not at all to the arbitrary (inertial) frames. Apart from such the exaggerated generalization of the result, this opinion may be also conditioned by some cosmic relations [3]. Owing to the cosmic expansion, two bodies are mutually moving away at the speed (v) proportional with their distance. Despite this fact, the light starts from its emitter, propagates through space and arrives to the detector at the same speed (c) – relative to the local surroundings. In fact, it propagates accelerating in relation to both mentioned devices: from the speed c up to c + v, or from c – v up to c, respectively. The apparent difficulty is thus exceeded.

Doppler’s effects, at motion of the signal emitter or detector (3), respectively, strictly obey the frames connected to the local media. The ratios of the propagated and emitted frequencies – in the former, or of the detected and propagatedones – in latter cases, accord with the ratios of respective speed pairs [3]. If the two relative speeds, (c – u) & (c – v), were equal to the absolute one (c), these two effects could not arise. At common motion of the two devices, the two effects compensate each other, with the detected equal to emitted frequencies. The same result arises at opposite motion of the medium, irrespective of the propagation itself corrected for the medium speed.

wc/wu = c/(c – u) wv/wc = (c – v)/c (3)

However, at light propagating through running water Fizeau obtained the result (4), dependent on the factor k, as the ratio of the material and total field components [1,2]. Apart from the slower propagation, the frame is drawn in the direction (v) of the water flow, as the moving material stratum.

c = co/er + kv k = P/D = 1 – 1/er (4)

The similar Faraday’s effect, twisting the polarization of light propagating along magnetic field lines, is caused by circulation of the static (F), in the form of kinetic (A) potentials. The former of them plays the role of the moving stratum.

A = emVF ∇·A = – em∂F/∂t (5)

The factor of the frame draw in the cosmic space may be similarly determined. Apart from the slower propagation in the present gravitation, the frame draw is determined by the ratio of the moving and dominant potentials.

Though applied in practice, the direct negation of the arbitrary orientation by Sagnac’s effect is ignored in the theory. Namely, two opposite light beams, propagating along the perimeter of a rotating figure, give the evident phase difference, according to the different relative speeds of light. This effect is multiplied by application of a solenoidal light conductor, instead of the figure perimeter. Such device is applied for registration of the angular airplane deviation, instead of the mechanical gyroscope. The references of the observer, in a resting laboratory or the rotating airplane, do not at all influence the result. This practical argument exceeds all the alleged relativistic proofs.

Transformations

The kinetic and dynamic inductions (1) added to the central static field [1] give the summary field around a moving charge (6). The longitudinal speed (V= v) of electric, and transverse (U) – of magnetic fields, obey the principle of relativity [1]. Alike the static field – at rest (E), the summary result (E’) is also centrally symmetric, tending to naught approaching the speed of light propagation. Apart from the magnetic field not affecting resting objects, the axial dynamic induction – subtracted from the radial static field, gives the ellipsoidal result.

E’ = E + (v – U) × B = (1 – emu2)E (6)

Missing the dynamic induction or understanding the object-field motion (u= v – U), H. A Lorentz formulated the summary force (7a). Expecting the similar and paralel magnetic phenomena [1], the symmetric magnetic relation was understood (7b), with the opposite mutual motion of magnetic poles and electric field. In fact, these two equations express the same relations, with the two equal and opposite mutual speeds. Not only that the free magnetic poles do not exist [1], but the field actions on the magnetic moments are essentially distinct. Apart from the former conditionalequation, latter one is fictional.

E’ = E + u × B B’ = B + emE × u (7)

The inverted set (8,9) exchanges the causes and effects. The opposite motion of the frame is understood. The set determinant (8b), as the factor (6) restricted to the transverse plane, points to the privileged frame, connected to the medium. For the sake of the frame equivalence adopted in advance, this value is arbitrarily distributed between the two sets: by 1/g in each of them. The formal inversion only is thus kept.

E = (E’ – u × B’)/g2 g2= 1 – emu2 (8)

B = (B’ – emE’× u)/g2 em = 1/c2 (9)

This action implies the explicit negation of vacuum medium at least. To push back the media, the product of the two constants is usually substituted by the speed c (9b).

By the factor distribution, the transverse field components (7) are arbitrarily increased, calling in question the mathematical form of Maxwell’s equations. Owing to their huge authority, the problem has been avoided by the complementary deformations of the two remaining 4D axes, longitudinal and temporalones!! (10). The former relation divides the position transformation (Fig. 4) by the factor g less from unit, but latter one also transforms the time. Owing to the limited relativity, these transformations are restricted to the uniform rectilinear speed u, in relation to the still unknown relevant reference frame!

x’ = (x – ut)/g t’ = (t – ux/c2)/ g (10)

Figure 4. The position transformation

The equations (10) somehow distinguish lengths and times in the two frames, depending on their mutual motion. Not only that this dependence cannot be physically explained, but lengths and times would depend on the speed, as their ratio! Moreover, the lengths depend on a given time beginning, as well as times – on the adopted frame position. Unlike the time possibly calculated from the frame overlap, the position of a chosen frame cannot be referred to any privileged location. The relative time, dependent on the object position – in the arbitrary frame, is undetermined!! Not only that the reference frames are arbitrary, but represent the mere mental constructs. There is not any logic by which their mutual motion can influence lengths and times, irrespective of any motion of the passive observers.

The ratio of the two equations (10) gives the relativistic speed transformation (11). Here v & v’ denote the object speeds in the two frames, and u – their mutual motion. The usual speed difference is here divided by the nominator less from unit, thus being artificially increased. Applied to light – as the object, this equation turns into the identity: c’ = c. Following through the sequence of inconsistencies, this is the pretext for the same speed of light at all (inertial) frames. This opinion is here already denied by the sequence of empirical facts.

v’ = x’/t’ = (v – u)/(1 – vu/c2) (11)

Avoiding or exceeding all these speculations, Hobble’s ratio of the distance and speed at the mutual motion away of two celestial bodies just understands the unique cosmic time. Some local times, deviating from the absolute one, are theoretically inconceivable and practically impossible. The real transformation of the position (Fig. 4), concerning the absolute time, is the only acceptable alternative. Namely, the positions of an object in the two frames differ no more or less but for the mutual distance covered after the initial frame overlap. In the unique space and time, this is the only reasonable relation.

General Relativity

Despite the sequence of formal inconsistencies above clearly presented, the restriction to a uniform rectilinear motion is the unique declared difficulty of special relativity. Alike inertia being generalized to the force action, Einstein tried to generalize his special to general theories. After some wander through chaotic speculations, he lost from view the initial problem. Owing to the kinetic and dynamic forces out of relativity, he returned to the static laws. On the basis of their possible balance, inertia is reduced to gravitation!? The radial field around a body is copied into the super-spherical space, along t-axis.

Alike gravitation projected on a slope, it itself is thus treated as the similar projection of some super-force acting from the cosmic center (Fig. 5). The field around a body is illustrated by the spatial curvature, as the local deformation of space, in the form of the funnel. At the interstellar space of the regular spatial curvature, such the projection equals to naught.

Figure 5. Local and global curvatures

Without any valid criteria, this concept is widely taken as the theory of gravitation. In the equivalent form, it speaks nothing about the essence of gravitation, but only further mystifies this phenomenon. If the formal mistakes and/or inconsistencies be excluded, there is no any reason by which its results will differ from the classical ones. At least accidentally, this analogy announces the radial cosmic expansion along temporal axis [3], irrespective of the red shift lately noticed.

Albert Einstein

In these times failing in the authorities of any kind, Einstein is an unquestionable scientific authority, if not in general, then in the modern physics at least. Instead of the resort to the praise, a brief and unbiased, as much as possible adequate valorization of his scientific contributions need be here presented. Avoiding unnecessary repetitions, the arguments rely on the above text and its references, also having in view the scientific criteria. Unlike majority of the other scientists, being famous concerning their unquestionable contributions, Einstein is celebrated with respect to his controversial views.

Apart from the photo-electric effect, Einstein deserved the leading position by the three ideas at least: the fourth dimension, curvilinear space and equation: w = mc2. Although the former two ideas were not originally his, Einstein emphasized and affirmed them self-confidentially. The indirect and complicated derivation of his equation seems to be accidental, resulted from the random formal procedures. Apart from the considerable simplification of this derivation, a direct its inference has been also presented in [2]. Not only that this equation is thus affirmed, but its convincing interpretation is finally enabled.

Unfortunately, none of the three mentioned ideas or results had been fully elaborated and applied by Einstein. At least in principle, the fourth axis is directly colliding with relative time. According to the tensor form of Maxwell’s equations [1], time is a real metrical axis. The probable restriction of the medium of light to the spatial gravitation excludes its temporal propagation. The relative time calls in questions its physical sense and reality. Not only that this concept is fantastic and arbitrary, but cannot be argued by any physical reason. At least some of its contradictions are here already clearly presented.

The idea of curvilinear cosmic space has been also unfinished. Though the Riemannian frame excludes the surrounding cosmic background [3], the modern cosmologists understand it. Even the surrounding cosmoses are expected in the background thus tacitly predicted. This inconsistency of the followers, not trying to finalize Einstein’s principal ideas, may be understood: the famous authorities would not make logical mistakes or give incomplete ideas. His equation has not been interpreted so far. Instead of its undefined application, it can be ascribed to the massive particles, unlike similar Plank’s relation for photons.

At least a part of Einstein’s fame relies on the arbitrary spatial orientation, as the main his failure. It implies the same speeds of light at all so called inertial reference frames, declared equivalent by the principle of relativity. Without sufficient vision of physical processes, as the corrective criterion, the formal calculations are prone to various mistakes or inconsistencies. In the final instance, the relativities of lengths and times are unacceptable at all. As the needed condition for these views, the vacuum medium had been explicitly negated. Imposed by the authority, this view still hinders the further development.

Let us also emphasize some Einstein’s methodological errors, mainly reducible to typical inconsistencies. Such is the arbitrary substitution of two similar math expressions by their geometric average. The average of two Doppler’s effects he applied to the cosmic red shift of light [3]. The opposite signs in the two field tensors he substituted by the imaginary unit [1]. In the opposite sense, the set determinant (8b) has been distributed between the two sets. The parallel EM phenomena (7b) were already understood. By the explicit negation of the medium, Einstein imposed the kinematical relativity.

The incomplete or doubtful principal views may be added. Apart from the static interactions, the relativity can be applied to the special cases of some distinct forces. Concerning two Mach’s ideas, Einstein chose the frame equivalence as the worst one. The unexplained kinetic induction was neglected. The lengths and times dependent on the moving observers fail in any explanation. The dependence of the relative time on the adopted spatial frame has not been noticed even by the numerous opponents!? The wide acceptance of relativity is not strictly founded, but imposed in the absence of the competent critiques.

By mess of the notions, Einstein confused the opponents. He substituted the two EM constants by the abstract speed of light, thus pushing back a vacuum medium at least. The absolute light propagation through the medium, irrespective of the instruments and observers, he substituted by the invariant relative speed. The temporal cosmic process, independent of the transverse spatial motion [3], he ascribed to the light propagation in a given spatial direction. The same speeds of light in the local cosmic situations imposed the additional confusion. All these facts taken together made the full mess in the theory.

Substitution of some theses is also frequent. The ellipsoidal electric field deformation around a moving charge, caused by the longitudinal dynamic induction [1], he ascribed to the increased transverse fields, by distribution of the factor g. The incomplete medium draw by the running water he ascribed to the relativistic speed transformation. Instead of the stronger structural stability, the shorter lives of speedy mesons are ascribed to the relative time. The two former cases at least do not obey the numerical relations. In the final instance, there cannot be even enumerate all Einstein’s inconsistencies.

Results

The relativity, as a postulate, is valid for the static forces only. Owing to various reasons, it cannot be applied to the kinetic and dynamic processes. The relativities of distinct physical forces are restricted to the special technical situations.

The same speed of light in a class of the frames, implied by the relativity principle and additional inconsistencies, cannot be anyhow understood nor explained. The generalization of the local to arbitrary orientation is exaggerated.

A sequence of the wave effects somehow contradicts to the relativistic views. At least one of them directly denies the arbitrary orientation of light. The navigational system (GPS) could not take into account the relativistic views.

The relativistic transformations are founded on the sequence of theoretical mistakes and inconsistencies. The dependence of the relative time on the adopted spatial frame is especially unacceptable and must be refuted forever.

Imposing relativity of lengths and times, the special theory lost from view the orientation. Reducing gravitation to geometry, the general theory did not generalize special one. These two theories only mystify the physical relations.

Not only that the restriction to so called inertial frames has not been exceeded, but its definition understands in advance a relevant reference frame. Going into the opposite direction, this opinion terminates in the local orientation.

Starting from the unique (absolute) reference – as the thesis, and arbitrary orientation – as antithesis, Mach’s local orientation in relation to the predominant material surroundings – as the synthesis, satisfies all the practical situations.

Conclusion

Between Earth and Sun, Copernicus chose the latter referent body. Newton similarly preferred the wider surroundings to the water container. Amongst the two Mach’s theses, of the local or arbitrary orientation, Einstein took the latter one. Not only that the orientation is thus overlooked, but disabled. The remaining local orientation, relative to a dominant mass, with some draw of the frame by other masses, satisfies all the practical situations. Although the rotation of Earth does not influence the satellites, its revolution firmly carries their orbits around Sun.

References

1. Miskovic BV. Foundation of Electrodynamics. Int J Phys Stud Res. 2023; 4(1): 98-103. doi: 10.18689/ijpsr-1000116

2. Miskovic BV. Unity of Physics. Int J of Theor and Math Physics. 2024; 2(1).

3. Miskovic BV. Curvilinear Cosmology. Int J Cosmol Astron Astrophysics. 2023; 5(2): 231-236. doi: 10.18689/ijcaa-1000143

4. Mišković B. A Brief Review of Special Relativity. Int J of Theor and Math Physics. 2019; 9(2): 36-40. doi: 10.5923/j.ijtmp-20190902.02

I am trying to clone a 1.3kb insert in a 3 kb vector. after double digestion of the vector, when I am checking on gel it is coming on desired length, but after ligation am getting almost same number of colonies in self (vector only) and test plates. after screening via colony PCR, none of the colonies had the insert. How to overcome this issue?

Hello, everyone

Do you know some techniques to improve the efficiency of reprogramming if I use a kit (cytotune-ips 2.0 sendai vector) expired in July 2021?

Thank you for your answers!

Hello everyone,

I am planning to do a cloning experiment. Can anyone suggest what is the minimum amount of insert DNA and vector DNA required for a 1:7 ligation reaction ? The size of my insert DNA is 673bp And that of the vector is 5.9Kbp.

Hello,

I am using movestay command for ESR model analysis, but I get an error message.

Can anyone help me please?

movestay (ln_wage = $x), select(union= $x msp) vce (robust)

The error message is:

Fitting initial values .....initial vector: copy option requires either a matrix or a list of numbers

r(198);

Thanks in advance for your kindness

Here's my issue--I've been troubleshooting cloning for the last 2-3 months but can't seem to get any colonies. I have been able to digest the plasmid (E. coli PLJR965 ~8600bp) at a reasonable concentration using BSMBI-v2. I want to ligate my vector with a ~25bp insert using T4 DNA ligase I believe the issue lies with the ligation reaction since my digested plasmid looks good on a gel. Here's what I've tried and hasn't worked to get my vector and inserts to ligate:

- PCR purification of digested plasmid before ligation

- Heat killing restriction enzymes for digested plasmid before ligation

- varying ratio of insert:vector (3:1, 10:1, 20:1)

- using new T4 DNA ligase buffer

- 30 min ligation at RT

- overnight ligation at RT

I've run controls with transforming just my undigested plasmid and that worked, so I know the problem likely isn't the transformation. I'm at a loss for what I can try next. Any suggestions appreciated!

I have two different construct:

1. Insert 1 (~3kb) at XhoI and ApaI site in NCVB vector (9.7 kb).

2. Insert 2 (~1.7kb) at XhoI and ApaI site in in pENTR/D vector (2.6kb)

I want to replace Insert 1 with Insert 2. I have already tried restriction digestion followed by Quick ligase (NEB) or T4 ligase (Thermo). I even tried using CIP as I got a large number of self-colonies without it. However, upon CIP usage, no colonies were seen in either plates (self and test). Kindly suggest ways to go about this cloning. The final product that I want is

PS: I am using ultra-competent DH5a (CSHL protocol) for all my cloning.

Hello, I am conducting a gibson assembly with 2 inserts and 1 vector.

vector size = 13kb

insert A size = 1.7kb

insert B size = 4.2kb

So, I tried gibson with 1:3:3 (vector:A:B) molar ratio.

Is this an optimized ratio?

I haven't tried it yet, but what about 1:7:3 (vector:A:B)?

Thank you.

I'm assembling one PCR fragment (4.2 kb) with my vector, pECFP (4.8 kb), and transforming them into NEB 5-alpha Competent E. coli cells. Both the fragment and the vector were purified using the gel extraction method. I have followed the instructions of the kit, trying different ratios of vector to fragment (1:1, 1:2, and 1:5) and allowing the reaction to proceed for 60 minutes or more, although there is only one fragment.

For transformation, I used 2 μl and 4 μl with 50 μl of NEB 5-alpha Competent E. coli cells for a heat transformation and then streaked the transformants on Kao plates (100 μg/μl). However, most of the time, I observe no colonies growing the next day. On the rare occasions when colonies do appear (only 7), they all contain the vector.

My next step was running 5 μl of the Gibson assembly product on an agarose gel to confirm the presence of the expected band (8.8 kb), and indeed, it was present. Despite trying various conditions, I consistently obtained a faint band, insufficient for successful colony formation.

Do you have any ideas on how I can improve my assembly and obtain the desired product? I would be very grateful, as I have exhausted my current strategies.

Thank you very much :)

I am planning to do Insitu-hybridisation. I have done the cloning finally . I cloned the PCR product in pGEMT easy vector . after doing the miniprep. I did the restriction digestion with sspI.

size of insert is 500 bp,

pGEMT easy vector- 3015

avaII cuts in insert at 11 positions and in vector at 1533,1755.

after running the gel I got the gel bands at 2000,1256,222 . and 1694,1562,222

which ORIENTATION I should consider for probe preparation.? how to do the linearisation? and How to select the T7 or SP6 polymerase for invitro transcription

Thanks

I need something like the image given below however with greater number of words and their associated works

Hello, I am trying to implement some boundary conditions in my model to simulate displacement currents "J" due to migration in an electric field.

According to the literature, I have to implement the following transport of charge carriers boundary conditions ( PLEASE SEE ATTACHED SCREENSHOT)

For example, for "p+", if the electric field multiplied by the normal vector is less than zero, the flux entering the solid should be zero. Otherwise, the flux should be nJ, where n is the outward normal vector from the liquid side, and J is the current flux given by upE, where u is the mobility, p is the charge density, and E is the electric field vector. I have been trying to implement this on the interface of the solid without success for at least 4 months. If anyone knows how to tackle this, it would be much appreciated

I have atomic coordinates and the structure is cubic cell with lattice parameter 10.001 Angestrum. How could I prepare the cell vectors to visualize in Vesta? Thanks.

I have inserted crispr vector in BL21 competent cell with ampicillin resistance and after induction with IPTG for protein ...no cas protein band was obtained using SDS page.. not even near to its size both in the supernantant as well as in the pellet... Can anyone help me.. the vector I ordered contains 6x his tag... But no protein after NI Nta agarose...

I ask a physical interpretation of the relationship between states of a quantum system introduced, in analogy with the causal relationship between events in Minkowski space-time by means of the formula xRy if and only if < x-y/ T( x-y)> > or = 0. Here x, y are vectors of a Hilbert space and T is a Hermitian operator applied in it. In the original relation x,y are Minkowski space-time four- vectors and T is the metric tensor diag ( 1,-1,-1--1). The relation is equivalent to the inequality <T>(x) + <T>(y) > or= 2 Re (<x/ T(y)>) where the first member is the sum of the mean values of the operator T in the states x and y respectively. ( Gennaro Franco, Giuseppe Marino, Possible causal relationship extensions and properties of related causal isomorphisms, Linear and nonlinear analysis, january 2020)

I digested my cas9 vector by adding 5ug of vector, 3ul of NEB 2.1 buffer, 3ul of BbsI enzyme and completed to 30ul with ultra-pure H2O.

I incubated overnight at 37C and added 1 ul of CIAP incubated for 10 min at 37C.

I ran my digested vector on an agarose gel and there is no visible band.

Although I can see a band for the intact cas9 vector, I don't for the digested one.

I had already made this digestion and I could see a band before, now it disappeared and I tried to make some new digested vector and there is no visible band as well.

I don't understand what is happening, if anyone has any idea.

I am trying to clone 2 genes from bacillus subtilis within a triple strong promoter ( amyQ, amyL,amyE) in E.coli ( both EPI400 and JM109 )with PUC57 and pgD1662 vectors, but I failed for several attempts. as no colony which including my construction survive. I want to ask if the triple promoter is leaky in E.coli and is there a problem with triple strong promoter? as its constitutive? (like changing the E.coli metabolism because of my genes expression?) or the construction ,includes triple promoter and 2 bacillus genes, is not stable in E.coli?

or do I need to change my E.coli strain? do you have any idea of how can I clone my full construction in E.coli? I did not find the problem yet. does anyone has the same experience in this area?

regards

I am trying molecular cloning of vector 5.717 kb with 3.792 kb of the gene with a single digestion. However, I am giving phosphatase treatment, but I am still getting a self-ligated vector after transformation. What could be the possible reason for it?

I am looking for exact copy number of pET28a plasmid. A citation would be great. Literature search only shows that it is low copy vector, but I haven't found any papers that mention the exact copy number or even an estimate.

Hello!

Recently, I ordered an FOLH1 ORF, which came inserted into the pUC-57 mini vector. Additionally, I requested BamHI and XbaI restriction sites to be included at each end of the ORF. When I performed the digestion of the plasmid, I observed that the single digestions with BamHI and XbaI linearized the plasmid as expected. However, when I performed the double digestion, I noticed two bands corresponding to the pUC vector and the FOLH1 ORF, as anticipated. Surprisingly, there was an additional band below them that I did not expect. What does this additional band signify? Could it be contamination, or is there a problem with the digestion process?

If we have to complement a vir gene in the Agrobacterium mutant, is it necessary to complement the vir gene promoter too?

I am planning to use pBBR1MCS-5 vector which has Lac/lac, T3, T7 promoters, are any of these promoters sufficient for my vir genes to be expressed in Agrobacterium mutant?.

Thank you in advance

Some context:

I'm trying to generate a clone possessing a gram positive phage endolysin gene in E. coli DH5alpha.

I'm using the pet28 vector and initially tried to clone/transform directly into both JM109(DE3) and pLysS with no success.

I can confirm the ligations are working with amplification of the cloning site.

I have even tried fusing the gene to GFP, constructing the pet28_GFP vector first and once I try adding this endolysin I fail to get clones.

Can a gene lead to complete plasmid instability in cloning strains that doesn't possess T7 RNAP which T7 vector systems rely on for expression? I am aware of T7 lysozyme repressing T7 RNAP and maybe there is a possible of this gene performing a similar role

I'm aware the protein is toxic but surely one should be able to generate a library to see it all go wrong in expression attempts.

Could my problems lie in DNA secondary structures effecting plasmid stability (replication)? any insights would be great.

<<The job is described in the followign code, where the gs.chk file is the result of a successful opt + freq calculation.>>

--link1--

%OldChk=8PhOPc_DMF_gs.chk

%Chk=8PhOPc_DMF_ex.chk

# td=(singlets,nstates=12) CAM-B3LYP TZVP Geom=check guess=read

scrf=(solvent=n,n-dimethylformamide) Int=(Ultrafine)

8-PhOPc TD 12 singly excited states: CAM-B3LYP/TZVP

0 1

<<This is the last few lines of output.>>

1 vectors produced by pass997 Test12= 5.85D-13 1.00D-09 XBig12= 5.26D-15 1.08D-09.

1 vectors produced by pass998 Test12= 5.85D-13 1.00D-09 XBig12= 5.35D-15 1.18D-09.

1 vectors produced by pass999 Test12= 5.85D-13 1.00D-09 XBig12= 5.23D-15 1.08D-09.

1 vectors produced by pass*** Test12= 5.85D-13 1.00D-09 XBig12= 5.29D-15 1.14D-09.

CPHF failed to converge in LinEq2.

Error termination via Lnk1e in /opt/apps/gaussian/16.c.01/g16/l1002.exe at Thu Jan 25 22:29:53 2024.

Job cpu time: 45 days 22 hours 11 minutes 56.9 seconds.

Elapsed time: 1 days 3 hours 47 minutes 34.0 seconds.

File lengths (MBytes): RWF= 224288 Int= 0 D2E= 0 Chk= 337 Scr= 1

I am trying to express a fusion gene using pet28a vector initially did a fusion by using SOE PCR method then RE digested the fused gene by using bam nd xho enzymes and ligated with pet28a vector and did a transformation to Top10 cells now where I am not getting the colonies I repeated the same work several times with positive control working fine and all my enzymes are working fine please anyone help me with this problem

Hello,

I tried to clone 3 insert into plasmid pUC18. I used Takara infusion cloning with this reaction:

1 μl 5X In-Fusion HD Enzyme Premix

0.59 μl Linearized vector (2667bp, DNA mass=44.35 ng, conc=179.1 ng/ul)

0.85 μl Purified PCR fragment I (3740 bp, DNA mass=62.19 ng, conc=175.4 ng/ul)

0.77 μl Purified PCR fragment II (482 bp, DNA mass=16.03 ng, conc=99.2 ng/ul)

1.84 μl Purified PCR fragment III (1694 bp, DNA mass=28.17 ng, conc=36.6 ng/ul)

In total I have 5ul reaction volume and do the infusion reaction for 20 minutes in 50 degree C.

I used NEB calculator to get my DNA mass. Is my calculation correct. I search in takara website they have their own calculator to count the DNA mass. Please give me suggestion is there something wrong with my method? I have been failed working with this clone for 3 months.

Thanks

Hi,

I'm facing a recurent problem with the cloning of a given cDNA. Everytime I'm doing the cloning I end up with a mix of big and small colonies on the insert+vector plate while getting only few big ones on the vector alone plate. The small ones are clearly not satellites, I know how do they look. I picked some of the small one but they are generally not growing in liquid. Tried to grow plates at 30°C no changes. Ran my ligation on gels and they look very efficient. I clearly don't know what to do. Any suggestions? Thanks

I am using a Tet on system to overexpress a LncRNA in mouse ES cells, based on a piggybac vector (pXlone). I performed transient and stable experiments. In both cases, the LncRNA is expressed even without adding dox in the culture medium. Can someone help me on this ?

Thank you very much.

Vector 1 (Insert 1, Insert 2, Insert a ) and Vector 2 ( Insert 1, Insert 2, Insert b) contain three inserts in which only the Insert a and b are different. These two vectors are constructed by Gibson assembly and transformed into E.coli DH5A cells. Positive clones are obtained based on antibiotic resistance. Plasmid was isolated for further cloning. While PCR check, Insert 1 &2 of vector 1 are amplified and visualised as a bright band, whereas when the same primers were used for amplification of Insert 1 and 2 in vector 1, it is lighter. The template concentration of vector 1 was also varied and checked.

Apart from primers specific to inserts 1 and 2, primer was designed for upstream and downstream regions of inserts. In that case, I am getting proper amplification for both vectors.

What could be the reason for getting different intense bands when the same primers are used for the amplification of the same inserts in different vectors?

Hi everyone.

I put a ligation reaction containing a 1 vector (~8 kb) and 2 inserts ( 434 and 537 bp) under bellow condition:

vector: 3' xbaI....... AgeI 5'

insert1: 3' xbaI ..... EcoRV 5'

insert2: 3' EcoRV ......AgeI 5'

ratio vector:insert= 1:7 (ratio: 1 vector+ 7 insert 1+ 7 insert 2)

overnight, 4.C

I have some colons but it seems that only one insert exist in the vector! how is it possible ?!

does anyone any suggession to can have the right transformed colons?

Also, does anyone have an idea how big your insert can be in a 6 kb vector when you use the gibson assembly as the cloning method to create a midigen and where you only design a forward primer at the beginning and a reverse primer at the end of your insert?

I have now developed a midigen with an insert of 4 kb (NF1 gene exon 20 to 24 with introns) in a pSPL3 vector of 6 kb, so together that is 10 kb. I did this using the Gibson Assembly cloning method. I designed a forward primer in intron 19 and the reverse primer in intron 24. For sequencing, I designed new primers to divide the 4 kb piece into pieces.

I am trying to develop midigene to study larger splicing effects with the mini/midigen exon-trap assay.

Hi, I am attempting to co-express GFP using a bicistronic vector that contains the T2A peptide.

(CD8 alpha signal peptide - GOI - T2A - GFP)

I am concerned that, since I am using the CD8a signal peptide, which directs the expressed protein to the membrane, GFP may also be directed to the membrane, making GFP expression undetectable in the cells.

Will GFP be localized in the cytosol if I use the vector I described above?

I look forward to your kind reply.

Thank you, and I hope everything goes well for whoever reads this.

I am using pPICZalfa vector with gene length 1.1 Kb. Currently I am trying for In-vitro multicopy generation (Bgl II - BamHI), but the transformation efficiency in DH5alfa is decreasing as my construct size is increasing. Should I try PTVA method?

I am using a pET28B vector to clone my 2.1kb insert digested with BamH1 (fwd) and Xho1 (rev) restriction enzymes. I'm using over 1ug of DNA for restriction digestion, kept at 37°C for over 3 hours. For cohesive end ligation, I'm using Promega T4 ligase, and keeping the setup (3:1, 5:1 and 7:1; insert: vector) for 16 hours before transforming in Dh5alpha competent cells followed by plating on Kan plates. I've been doing this for quite sometime now. But everytime, all I get are false colonies. I've tried to troubleshoot every single step but to no avail. My ligase is also new.

Hello!

I'm trying to see if anyone has a PDF of this paper, "Factors Influencing Lentiviral Vector Quality-Dynamic Light Scattering (DLS) Analysis of HIV-1 Vectors"

I would appreciate it if you can send it to me.

Thank you

1. My overexpression plasmid (empty vector) is 8200 bp. But it is showing 4000-5000 bp always in the agarose gel, which might be for the supercoiled conformation. But this large difference is acceptable?

2. The same plasmid after digestion ( EcoRI & BamHI) becomes 12000+ bp in the agarose gel. Shouldn't it be around 8000-8200 as it becomes linear after digestion? If it becomes 12000+bp which is too big, is this acceptable? and why it becomes bigger.

My colleague is trying to study the effects of a protein on pathogen inhibition in N. benthamiana. He is expressing it in either a pSITE vector or pCAMBIA1302 that had the GFP gene removed. They are both in GV3101 strain of agrobacteria. His protein is being expressed fine and he got interesting results. However, when he infiltrated just the vectors as controls, the pCAMBIA gave a similar response as his protein but the pSITE did not give any response. A response was not seen in the no infiltration or buffer only conditions. Has anyone else found that the vector backbone alone can alter plant response?

Time dilation has absurd predictions. So, it may be an illusion. Is it possible that this mistake has its roots far back in 1887, when Michelson did his famous tests? The interpretations of these tests assumed that the mirrors in the equipment define the vector sum of ether wind v and wave vector c. This was a reasoning based on particles, and the mirrors have relevance only in relation to c (but not to v), and the wave model is in conflict. So, time dilation appears to be an illusion, and the Lorentz transform not needed.

Einstein gave support to this idea by postulating that observers in different (but constant) motions all see the same light speed, but Einstein's idea is also absurd.

Hi!

Has anyone worked with negative-sense RNA viruses? I need to express recombinant proteins using a vector, so it will be necessary to start with ssRNA(-) to obtain cDNA for selecting the gene of interest. Are there kits with RNA-dependent RNA polymerase to obtain the positive-sense strand (necessary for the following step with the reverse transcriptase), or do I need to isolate the enzyme in some way? Thank you.

Actually I'm trying to express my protein using ppiczaplha series of vector in x-33. My transformation is successful as I'm getting two bands after amplification through aox1 primers from genomic DNA. I have tried different parameters for expression like different temperature(20-30), different methanol concentration, expression protocol for Mut+ as well as Muts, i got expression once after that result is not reproducible. I don't have protease-deficient SMD strains, but we have pichiapink strains that are protease deficient, so can i use this?

Hi, I just have been in fusion cloning.

I want to insert my GOI in pMIG II vector.

So, I amplified my GOI using PCR with 15bp overhang (PFU used), and linearized the pMIG II vector with EcoRI and XhoI.

(PCR fragment and linearized vector were cleaned up using gel elusion kit)

I used Stbl3 for transformation.

And I used in fusion molar ratio calculator to determine the DNA concentration of fragment (1728bp) and vector (6512bp).

But, I failed to get plasmid with my GOI. and I sequenced this failed plasmid and found that this plasmid matched the sequence of the intact pMIG II.

(E.coli did not grow in linearized vector only control)

How do I solve this problem?

Hello everyone,

I save my FEM results as VTK files, these files include data such as Points, Variables values, mesh type etc.

I have been trying to get a vector graphic (like SVG) to display smooth and nice results.

I tried ParaView, but I think they do not support vector graphics in recent versions.

I also tried to write a Python code for this purpose using vtk and matplotlib libraries. It works almost fine, but when I want to plot the mesh too, there are problems.

I used Triangulation from matplotlib.tri, but it only supports triangles mesh, while my mesh type is 9-node quadrilaterals.

So, the question is, what is the best way to get SVG image of a VTK file?

Thanks,

Masoud

I generated Hela-KRAB-dCas9 stable cell line, and to validate dCas9 activity, I introduced two sgRNA expressing vectors driven by either hU6 or mU6 promoter. I found that the mU6 promoter driven vector did show any knock down effect, but the hU6 one did. I wonder if mU6 promotor is really inactive in Hela cell, and whether mU6 promoter can be active in some human cell lines. Any one has this information? So many thanks!!!

I would like to ask a question about constructing stable cell lines.

If someone has the whole genome sequencing results of their overexpression stable cell line that would be really helpful. That would give us a clear solid example of what going on during the fregment integration steps.

Thank you for your reading, and please let me know if I did not make my idea clear. It would be really helpful if you answered my question.

Best,

Le

Aedes mosquito species, i.e. Aedes aegypti and albopictus, can act as a vector for both Flavi viruses (Dengue serotype 1-4, Zika virus, West Nile Virus etc.) and Alpha Viruses (Chikungunya virus). What is the mechanism behind this and is it possible for the Aedes mosquito to hold both Flavi and Alpha viruses at the same time?

Please share if you find any articles related to this. Thank you.

I am looking for vectors for expression of bacteriocin, and from pET series I think that 19b fits the most, I am just having few questions. When I clone my insert in NdeI and XhoI, i will have residual H in N-terminus, which I don't want. How can i avoid this?

Also, can i put stop codon at the end of my insert, will that be sufficient, or should i add more stop codons?

I want my protein to be without residual (from vector) amino acids on N and C termini.

I am open for all suggestions regarding the choice of vector, since i can't get expression in pQE and pMAL vector using M15 and ER2523 E. coli expression cells.

In attachment is pET-19b from Genscript.

In fuzzy set theory, while handling multi-attribute decision making problems, there are few conditions imposed on weight vectors (weight vector for attributes, for experts), that each component of these vectors must be greater than zero, taken from [0,1], and the sum of their components must be equal to one.

I require the empty vector of this plasmid: https://www.addgene.org/40346/

The ends have been destroyed:

How do you suggest to do this?

Hello everyone,

Is there anyone who has cloned with pAcGP67A secretion vector with GP67A signal peptide.

I tried to clone with insert 1.2kb in the vector, MCS: BamHI and NotI. C-term Thrombin and His-tagged was engineered.

I keep getting a stop codon after the signal sequence before the BamHI then my insert at the N-terminal.

I simulate with Snapgene but i keep getting inframe with GP67A and a stop codon appears .

Please i need a help.

Thank you

human Topoisomerase is a HOMODIMER Protein (AA). Now i want to mutate one chain to produce a heterodimer version (AA*). but there are chances of production of three products (AA, AA*, A*A*). So the question is that how i should place the two Genese wild type (A) and mutant (A*) in E coli expression system that only one required form AA* is produced?

What is the difference between vector pET (26,21, a,,b,+.........?

What are the changes made in the PET vector (for different pET)?

In B-spline interpolation, how control points and knot vector influences on trajectory optimization for robot path planning?

Hi, please i need a help. Attached is my Agarose gel containing ligation reaction (pET28a) and VEGFA (insert).

After ligating for overnight at 16 degree and run gel with 10uL from the ligation reaction.

Here is what i got.

Lane 1: Uncut vector pET28a

Lane 2-3: VEGFA insert 1

Lane 4-5: VEGFA insert 2

What could that suppose to mean?

Thank you.

I have been trying to digest a vector pXMCS for the last two months but no luck so far. I have been using NdeI and KpnI (Both of them were present on the plasmid map of the vector) as restriction enzymes and incubating the reaction at 37 degrees for 5 to 6 hours. I am using buffer 2.1 which is compatible with both enzymes. Even overnight digestion does not seem to work out. I also tried single enzyme digestion reactions for 5 to 6 hours and overnight (just to check which one of the enzymes is not working) but this is what I got (2nd picture). I would love to get some suggestions on the same. Thank you.

PICTURE 1: Lane 1: 1 kb plus ladder, Lane 2: uncut vector, Lane 3 and 4: Digestion reactions (with 1ug concentration vector each), Lane 5: Uncut vector, Lane 6: Digestion reaction (3ug vector)

PICTURE 2: Lane 1: 1 kb plus ladder, Lane 2: uncut vector, Lane 3: KpnI, Lane 4: NdeI

I am using Gibson assembly method for insert my PCR product with the length of 3.6 KB into a vector, I excise the original vector to remove unwanted gene with specific restriction enzymes and use HIFI DNA ASSEMBLY for insertion PCR product into the vector, after assembly I used it for transformation finally miniprep, and for final approve of my work again use a enzyme that cut my plasmid exactly in the middle of the inserted gene, but the result on the gel is not satisfactory. what can I do?

My colleague has come up an idea of using two inserts to ligate into one vector at the same time, which is like : vector-SpeI-Insert A-BamHI-Insert B-NotI-vector

(Spel-Insert A-BamHI, BamHI-Insert B- Notl, Vector digested with Spel and Notl).

Then he transformed the system into E.coli and today the colonies showed up.

Unfortunately, the verification PCR only shows the band of Insert B fragment.

(vector is around 3kbp, Insert B is around 1.8kbp, Insert A is around 700kbp, the verification PCR's predicted size is around 2.5kbp containing the part of Inert A and B) We are confused about the result and I really wish to hear from your suggestions.

Are electric field vectors always perpendicular to equipotential lines why or why not and equipotential surfaces are not equidistant for a point charge?

My colleague has come up an idea of using two inserts to ligate into one vector at the same time, which is like : vector-SpeI-Insert A-BamHI-Insert B-NotI-vector

(Spel-Insert A-BamHI, BamHI-Insert B- Notl, Vector digested with Spel and Notl).

Then he transformed the system into E.coli and today the colonies showed up.

Unfortunately, the verification PCR only shows the band of Insert B fragment.

(vector is around 3kbp, Insert B is around 1.8kbp, Insert A is around 700kbp, the verification PCR's predicted size is around 2.5kbp containing the part of Inert A and B) We are confused about the result and I really wish to hear from your suggestions.

Is magnetic field always perpendicular to electric field and what are the electric and magnetic field vectors in electromagnetic waves?

I downloaded the starlink precise ephemeris file from https://www.space-track.org/, the data of each time point includes position, velocity vector and coordinate data. May I ask which coordinate system the vector is in, and what is the meaning of the 21 covariance value

Hi dear researchers

I need to calculate the insert mass for the ligation reaction, the vector length is 7800bp and the insert length is 83bp, would you please guide me what is the correct and suitable insert/vector ratio? my insert is very small in comparison to vector

I used 10;1 , but I did not obtain my results.

I appreciate you for taking the time.

I'm using a transposon tol2 vector (pT2AL200R150G - 10.1534/genetics.106.060244) to create transgenic zebrafish line.

We first inserted our gene of interest (GOI) into the BamHI site of the vector, which was fused with the eGFP sequence already present in the plasmid. Next, we replaced the native promoter of the vector (ef1a) with another promoter by digesting the vector with XhoI and SalI and ligating the insert via in-fusion ligation.

After cloning and validating, we injected the vector along with the transposase mRNA into the one-cell stage zebrafish embryo. The following day, we observed GFP expression, indicating expression of the GOI fused to it, mainly in the vector with native constitutive promoter Ef1a+GOI. However, after a few days (3/4 days), the fluorescence expression gradually disappeared until it was no longer visible. This is unexpected, as we injected transposase RNA, which should theoretically insert the transposon into the animal's genome. We are wondering if this is a normal occurrence and whether crossing F0 will result in expression in the offspring.

P.S. We performed PCR on the injected animals for both GOI and GFP, and although we observed amplification of the sequences, we did not detect any expression.

1. My overexpression plasmid (empty vector) is 8200 bp. But it is showing 4000-5000 bp always in the agarose gel, which might be for the supercoiled conformation. But this large difference is acceptable?

2. The same plasmid after digestion ( EcoRI & BamHI) becomes 12000+ bp in the agarose gel. Shouldn't it be around 8000-8200 as it becomes linear after digestion? If it becomes 12000+bp which is too big, is this acceptable? and why it becomes bigger.

Hello,

I'm exploring different modulation types like MPSK, QPSK, and MQAM in various environments. Currently, I'm measuring the error rate but want to understand and measure the Error Vector Magnitude (EVM) more accurately. I'm using a general equation for all modulation types. Is this the right approach, and what should I expect from the EVM vs. Eb/No curve? I'd appreciate any help.

I have insert having flanking regions with XbaI site and and a vector digested with XbaI. How can I prevent self ligation in insert as well as vector before ligation?