Vectorization - Science topic

Hi Sanjay T D I'm currently trying to clone a 700bp insert into a 18kb vector and I'm having the same issue as you : no colonies. I'm pretty sure the problem comes from the transformation part and not the gibson assembly. I've read a lot about it and apparently 18kb is really big for bacteria if you're doing a heatshock (like I do). people recommand using electroporation instead (if you can).

If you manage to clone your insert please let me know because I'm really struggling. So far I've tried different ratios 1:1, 1:3, 1:10: 1:20 with vector quantities from 100ng up to 300ng.

Have a nice day.

I am assuming when you say "it is not happening" means you are unable to see the 400bp insert in the gel? You do not say how much plasmid you digested but more than likely there is not enough insert in the gel to visualize.

Your 400bp insert is ~12% of the overall 3400bp size of the plasmid so if you digested only 100ng of plasmid, you would only have ~12ng of insert to see in the gel which is on the low end of what is possible to see in an agarose gel. If you double digest 500ng of the plasmid you should be able to easily see the insert (~60ng).

Thank you Robert Adolf Brinzer and Saman Behboodi Tanourlouee for your suggestions!

Sam

DH10Bac and DH10EmBacY cells are both used for the generation of recombinant baculoviruses for protein expression in insect cells. However, they have different characteristics and uses.

DH10Bac cells are used in the Bac-to-Bac system developed by Thermo Fisher Scientific. They are used to generate recombinant bacmids containing your gene of interest in a baculovirus genome. These bacmids can then be transfected into insect cells to produce recombinant baculoviruses.

On the other hand, DH10EmBacY cells are part of the EMBacY system, an alternative system for generating recombinant baculoviruses. This system also involves creating recombinant bacmids, but the process and the specific cells used differ from the Bac-to-Bac system.

In general, you should follow the protocol and guidelines provided for each specific system and use the recommended cells for that system. Interchanging the cells may not produce the desired results, as each system has its own optimized conditions and requirements.

Yes, if the transfective vector from the lentivirus packaging system contains a functional GFP gene and is successfully transduced into the target cells, the GFP gene should express and result in the production of green fluorescent protein (GFP) in those cells. This expression can be used as a marker to indicate successful transduction and expression of the transgene in the target cells.

Hi Haris

What tool do you use for DFT calculation?

If you use QUANTUM ESPRESSO then you have two options 1. use the xcrysden software and other is 2. use tools on QUANTUM ESPRESSO's official site.

With Regards

Sahil Dani

There are a few rules to design primers for gene cloning. If you follow them, there will be no issue. The rules can be easily get from PubMed. For in silico gene cloning, you can use the software SnapGene and ApE plasmid editor.

The error message you're encountering, "Error [65]: Error, extent of vector too large or attribute table error," typically indicates an issue with working with vector data (like shapefiles or other spatial formats) in a Geographic Information System (GIS) software like QGIS. This error can occur for various reasons. Here are a few things you can try to troubleshoot and resolve the issue:

If none of these steps resolve the issue, you might want to seek assistance from the QGIS community forums or support channels. Providing specific details about the dataset you're working with and any additional error messages you encounter can help others provide more targeted assistance.

Based on the information provided in the description of the CD3-434 vector on the Arabidopsis Stock Center website (https://www.arabidopsis.org/servlets/TairObject?type=stock&id=313445), it states that the vector contains the Kanamycin resistance gene, which confers resistance to Kanamycin in plants.

Since you are working with Escherichia coli (E. coli) MC1064 as competent cells, you would need to use an antibiotic that is effective for selection in bacteria. Kanamycin is commonly used as a selection antibiotic in E. coli, so you can continue using Kanamycin for your bacterial selection.

However, it's important to note that the CD3-434 vector was originally designed for use in plants, and the information available does not specify whether it has been tested or optimized for use in bacteria. Therefore, it's advisable to verify the compatibility and functionality of the CD3-434 vector in E. coli by consulting relevant literature or contacting the Arabidopsis Biological Resource Center (ABRC) for further information or guidance.

Further more:

In addition to PubMed, Google Scholar, and Scopus, you may find the following databases useful for your search:

1. Web of Science: Web of Science is a comprehensive research database that covers a wide range of scientific disciplines. It includes a vast collection of scholarly articles, conference proceedings, and citation information.

2. Embase: Embase is a biomedical and pharmacological database that covers a broad range of topics. It is particularly useful for drug-related research and pharmaceutical studies.

3. IEEE Xplore: IEEE Xplore is a database specifically focused on engineering, computer science, and related fields. It includes a wealth of research articles, conference papers, and technical reports.

4. ACM Digital Library: The ACM Digital Library is a database dedicated to computer science and information technology. It contains articles, conference proceedings, and other resources from the Association for Computing Machinery (ACM) and affiliated organizations.

5. ScienceDirect: ScienceDirect is a leading full-text scientific database that covers various disciplines, including life sciences, physical sciences, social sciences, and more. It hosts a large collection of journals, books, and conference proceedings.

6. arXiv: arXiv is a preprint server that primarily focuses on physics, mathematics, computer science, and related fields. It provides early access to research papers before they undergo formal peer review and publication.

Tailor your search terms to the specific research topic or keywords related to the CD3-434 vector and its compatibility in E. coli. Exploring multiple databases can broaden your search scope and increase the likelihood of finding relevant literature.

Good luck

credit AI tools

Get help from the AddGene site to choose the right vector

Thank you Subham Basak

Hi Timothy K. Craig

There could be several reasons why you're not achieving the same transformation efficiency as the paper. Here are a few things to consider: Quality of Plasmid DNA: Ensure that your plasmid DNA is of high quality, pure and not degraded. Use a reliable method to isolate and purify your DNA. Competency of Yeast Cells: The competency of yeast cells is critical. Make sure that the cells are appropriately prepared, not too old or too young, and handled gently during the preparation process. Plasmid DNA Concentration: The amount of DNA used can impact transformation efficiency. Verify the concentration of your plasmid DNA using a reliable method such as spectrophotometry or Qubit fluorometer. Transformation Protocol: Be sure you're following the protocol closely. Small details, like the temperature and timing of heat shocks or the voltage and cuvette gap in electroporation, can make a big difference. Media and Growth Conditions: The choice of media, pH, temperature, and even the type of agar can affect transformation efficiency. Strain Variation: Different yeast strains can have different transformation efficiencies, even with the same plasmid. Make sure your yeast strain is identical to that used in the paper. Experiment Reproducibility: Even when a protocol is followed precisely, achieving the exact same results can be challenging due to variability in conditions and materials.

Dear Doan Cong Dinh,

Thank you for your professional input, and I appreciate this valuable article.

I will try to grasp the concepts of quaternion analysis.

To convert magnetic field data from a satellite dataset into the mean-field aligned coordinate system (MFAC), you'll need information about the mean magnetic field at the location and time of interest. The MFAC system is commonly used in magnetospheric and ionospheric research to express magnetic field measurements relative to the average magnetic field orientation in a given region. Here's a general outline of the steps to perform the conversion:

dear Ahmed Elsherbini to point 1: your concentration is fine. You don't need more than that. In general people use 100 ng of vector for a ligation IN TOTAL (that is about 8 ul of your purified fragment of 12ng/ul, which you should have; but you can also use consideraby less, it depends how efficient your cells are for transformation). Point 2; the 3:1 ratio vector to insert is NOT about ng but about molar ratio. that means 3 molecules insert vs 1 molecule vector. Your vector is 6kb, your insert 1 kb, so for 100 ng vector you need about 50 ng insert. So in summary: probably u use way too much insert. I don't know what u mean about the end product in electrophoresis? After transformation and miniprep? Maybe you get vector dimer, it can happen. As Anatoliy said, try dephosphorylating the backbone vector prior to purification. If after fixing the ratio, problem remains, make sure the sites in your PCR fragment are properly cut (enough base pairs at end next to restriction site etc).

Lo Tzu Yu This is the most likely cause of the reduced viral titer. Especially if the combined lenght of Ca9 GFP Puro and sgRNA cassettes exceeds the cargo capacity of the vector. To measure this, confirm that the DNA region between 5' and 3' LTR is below 8.5 kb. Bigger size will cause a reduction in viral titer and in my experience I haven't been able to package anything with a lentivector above 10 kb.

If you want to restore the size and keep eGFP and Puro, you might have to use a shorter promoter or a different (smaller) Cas variant, like CasX for instance. The latter will require also changing the sgRNA expression cassette (scaffold and spacer).

Hope it helps

Francesco

Valery Aleksandrovich, I sent this paper by a personal message.

You can try to look up recombineering methods. You don't need to cut the vector but you will need longer homologous overlaps.

Let me clarify the question of the compactification of the Minkowski space. As a result of compactification, the isotropic cone of the Minkowski space turns into a Euclidean space, to the zero point of which a three-dimensional sphere is glued. For clarity, you can reduce the dimension and imagine the plane on which the ball lies.

I would highly recommend testing it, if your current cells have it then it is possible also the stocks have it, which in turn imply whoever sent the stocks might also have it. That is what happened to me, we found out all our stocks were contaminated even for different unrelated cell lines which came from another institution. We used to following kit for detection though you can find other kits that will do the same: https://bioscience.lonza.com/lonza_bs/TR/en/Cell-analysis/p/000000000000186472/MycoAlert-Mycoplasma-Detection-Kit-%28100-Tests%29

Then we treated the cells to get rid of the mycoplasma, we even tested the stocks in liquid nitrogen and found out that it didn't start in our lab, we just bought it over from the other institution where the stocks came from! It took much effort but we overcame it. Also if your tests become positive, consider throwing everything out and starting over instead of mycoplasma elimination. We really spent a lot of effort with testing & treatment to re-establish our stocks. But in the end, we boosted our transfection efficency from 5-10% to 40-60%. There are many kits for mycoplasma elimination, here's one from Lonza if you decide to stick to their detection kit also:

To get all the information about your plasmid, it is better to use the https://www.addgene.org/ site

I hope you find a more complete answer in the following article,

Is it time to reformulate the partial differential equations of Poisson and Laplace?

Researchgate, June 2023.

Dear Brandon Rosales

each protein as its own history that depends from its specific properties as:

- MW;

- isoelettric point;

- presence of cisteines and or hidrofobic regions;

- presence of rare codons (expecially for protein derived from eucariotic)

so a protocoll the work well with some proteins can not work at all for some others.

Can you provide some more details about your specific protein?

From your message, i do not undestand if the problem is at the level of

- expression; (no overexpression band observed in SDS page total extract)

- solubility (no soluble band are observed in the soluble fraction but a band is observed in the total exctract)

- purification (a band is detectable in both total extract and soluble fraction but you are not able to enrich and purify it with IMAC and/or GST purification.

best regards

Manuele

It depends on the plasmid, and the promoters it contains. I have done this and got it work well enough.

search for bicistronic plasmids in addgene

You are missing the polybrene step; adding polybrene 1:1000 to your cells it dramatically improves transduction efficiency in the difficult-to-transduce cells. Also, for the LC3-RFP plasmid, you don't need stable transduction for autophagy evaluation experiments, and if your target cells permit, you can get away with transfection of the construct.

Hi there,

Your question is a bit of unclear to me... When you mention enzyme production, do you mean the enzyme produced by the gene conferring resistance or do you mean the product of the gene of interest possibly cloned into the vector?

Conrad Hübler

Understanding the differences in frequencies obtained from a numerical hessian calculation when the Eckart equation is not included is an important aspect of molecular modeling. The Eckart equation is commonly used to transform the mass-weighted Hessian matrix into normal modes by accounting for the rotation and translation motions of the molecule. Not including the Eckart equation can lead to several differences in the frequencies:

1. Translation and Rotation: The Eckart equation accounts for the translation and rotation of the molecule as a whole. Neglecting these terms can cause the frequencies to include contributions from these global motions, resulting in spurious low-frequency modes that do not represent molecular vibrations.

2. Zero Modes: Zero modes correspond to the translation and rotation of the molecule, and they should have a frequency of zero. When the Eckart equation is not included, these modes may have non-zero frequencies, leading to erroneous results.

3. Inaccurate Frequency Scaling: The Eckart equation is also used to scale the frequencies obtained from the Hessian matrix to match experimental values. Neglecting this scaling can result in frequency values that are not in good agreement with experimental data.

4. Numerical Noise: Numerical noise can also contribute to differences in frequencies, particularly for larger molecules. Small errors in the calculation of the Hessian matrix and subsequent diagonalization can accumulate and lead to deviations in the frequencies.

In your case, the presence of imaginary frequencies for structures in geometrical equilibrium could indicate issues with the optimization procedure or potential instabilities in the geometry. It's important to ensure that the optimization is performed correctly, considering convergence criteria and appropriate settings for the optimization algorithm.

I would recommend reviewing the implementation of your code, ensuring that the Hessian calculation, mass weighting, diagonalization, and unit conversions are correctly implemented. Additionally, consider checking the accuracy of the employed optimization algorithm and potential issues specific to larger molecules.

For further insights, I suggest consulting literature on numerical hessian calculations, vibrational analysis, and molecular modeling methodologies. Comparing your implementation with established methods and validating it with benchmark systems can also help in identifying and resolving any discrepancies.

The ‘writewa’ last command suggests your error occurs during a read/write to a word-addressable (wa) dataset. Hence, the error may be related to a lack in your storage device or - less probably, I suppose - to a disk error.

Hi! I'm having the same problems when trying to ligate the annealed oligos with lentiCRISPR plasmids. So do you finally find out what the problem was? Is it a plasmid problem or the problem is related to another component in the ligation mix?

According to my knowledge, the most appropriate method to access the accuracy and reliability of gene cloning is DNA sequencing.

The risks associated with gene cloning include nucleotide deletion that can be deleterious for gene function and the substitution of a few nucleotides with others that depends on the fidelity of the DNA polymerase used for the amplification of a particular gene.

There must be several software available for this purpose but I prefer to use SnapGene and ApE.

In addition to the typical pairs of compatible ends like Xho1-Sal1 or BamH1-Bgl2, all blunt end restriction are of course compatible, or any pair of sites can be rendered blunt and then ligated.

It is possible that two plasmids co-exist in the cell, so restreaking will not solve it. My suggestion is to dilute a little of your DNA and retransform it at very low DNA concentrations and then try a few of those colonies. It could be they are nearly identical but maybe slightly frame shifted, or you have some vector alone contaminating the actual clone.

The other possibility is that there is some secondary structure in your plasmid so you are getting some frame shifted sequence after the secondary structure.

Designing specific primers for the ptdT-RNAi vector in plants requires careful consideration of several factors. Here are some guidelines to help you design effective primers:

Remember to consult the literature and resources specific to your target gene or plant species for any additional guidelines or recommendations.

Finally, we succeeded in cloning. We confirmed that the problem was not in the ligation ratios, with the positive PCR results of the colonies formed at the ratios of 1:1, 3:1, and 7:1. The problem was in our enzyme. We have overcome this problem by using the new NEB T4 ligase enzyme.

Yes, I don't think I have the wrong type of plasmid either, as the plasmid has been used before without any problems by my supervisor in prior years.

If the vector without insert is showing the same band then either your vector-only culture is contaminated, or more likely, the band you are looking at is just a background host protein band and not the product of your clone.

Thank you Didier,

thank you, you make very good points.

I was considering to use oligo dT primers to ensure to clone full size transcripts. Therefore I hope that the adaptor in the 5' end of the transcript ensures the fusion of the Gal4 AD domain in all three frames.

I asked already Takara how they designed the system.

Thank you and best regards,

Janos

It would take time but you can look in the literature to see what vectors have been used in your species of interest. If they are uncommon to transform this won't take too long. If they are common species, there are probably commercially available vectors such as pCAMBIA or similar. The challenge here might be finding a selective agent that works well for all your species and cultivars.

The mass of the vector and insert are not the relevant parameters, what is relevant is the molar concentration. So if you have similar mass of plasmid that is 5kb and insert that is 0.5 kb then your molar ration of insert to plasmid would be 10:1 when the optimal is more like 2:1 or 3:1.

But with regards to volume, in most cases the more concentrated the better. If you have too little DNA then you may not get good inter-molecular ligation.

In the end, the people who gave me the R. rhizogenes (strain K599) did not know it was contaminated with an unknown bacteria that had taken over the original culture, so that is why the pVS1 ori was not working. Just for those who are looking for answers about the compatible oris, R. rhizogenes, and A. tumefaciens strains are compatible with the following origins of replication: RK2, pVS1, and pRIA4.

Cargos of >8kb result in reduced titers of virus. Consider switching over to a CPP functionalized filamentous phage system for really large cargos.

No, it does not need to be blunt. But you do need to understand what's left of your sticky ends after T5 nuclease is done with it. Draw the sticky ends in double-stranded notation, and then visualize how T5 nuclease is going to chew one strand back, but not the other.

Then, you can use NEBuilder to check on your understanding. It does it automatically.

can we open a set file only ?

sorry, D=a*(z1*z1+z2*z2+z3*z3)/sqrt(z1*z1+z2*z2+z3*z3) is the box size. The equation should be D=a*sqrt(z1*z1+z2*z2+z3*z3) / (z1*z1+z2*z2+z3*z3) / 2sin（A/2）where A is the misorientation angle

Hello, May I know how you resolved your issue? I am also facing same problem but in my case the enzyme I am using is Dam and Dcm sensitive. I am suspecting I should grow them in a specific bacterial strain before isolating plasmid. Thank you Shiva Khoshkhoo

What is the size of the vector plasmid? Also, what are your current titers?

When it comes to teaching Stokes' Theorem and its applications in CEng engineering, there are several methodologies and tools that can be utilized to make the learning experience more engaging and effective. Here are a few examples:

References:

Hanif Ullah

Use the lentiviral vector system; it works really well. Don't worry about the efficiency; you can select based on antibiotics available with the plasmid. Just be careful about starting antibiotic selection from lower concentrations and increase it with every passage.

Pscalps Puromycin or hygromycin can be requested from addgene. Please let me know if you need more details.

All the best.

Hi Robert Adolf Brinzer Thanks for the suggestion. I've industrial experience working with CHO platforms for biosimilar production. My PhD work also revolves around similar lines. I'm trying to study the factors responsible for enhanced expression of recombinant antibody, with respect to host cell line engineering as well as vector optimization. Vector optimization include use of chromatin-modifying elements and developing a mammalian expression vector. Also, we are trying develop a high efficient host cell line (CHO) which when used can produce higher yields.

ρ = Σb/2πh

Where ρ is the edge dislocation density, Σ is the total length of dislocations per unit area, b is the magnitude of the Burgers vector, h is the interplanar spacing of the diffracting planes, and π is the mathematical constant pi.

Assuming that we know the interplanar spacing of the (10-12) diffracting planes, we can rearrange the equation to solve for the Burgers vector:

b = 2πhρ/Σ

So, to determine the magnitude of the Burgers vector, we need to measure the edge dislocation density using (10-12) XRC and the total length of dislocations per unit area in the material. We can then plug these values into the equation above to calculate the magnitude of the Burgers vector.

Yes, you can use Octave, Maxima, or SageMath for computing the quadratic equations coming from the Jacobi identity for a vector space equipped with a bilinear, skew-symmetric bracket.

Here's a brief overview of how each of these software tools can be used for this task:

All of these software tools are available for Linux, so you should be able to use any of them for your computations.

First of all, the strategy in B is only for bacteria and not eukaryotes unless you introduce specific signals like an IRIS signal. Secondly, B only establishes similar transcription for the two genes, but not necessarily similar translation. So it is unlikely that either will guarantee precisely the same amount of both proteins, although admittedly B is likely to be more similar. The main advantage of C is that it may be easier to make the construct, and easier to for example make a bunch of mutations in one and not the other.

Often you don't need the same amount of both proteins unless it is a heteromultimeric protein. But both approaches do work and the choice really depends upon the precise goals and details of the projects.

Not sure what exactly might be the reason. Mostly has to do with structural differences between the two.

You can go through the following articles which might give you a better idea.

1) Discussion section of

2)

Try increasing the template concentration of the circular plasmid and see if you are getting non-specific amplification.

Thank you for sharing your experience dear Malgorzata

Hi Elina,

I just saw your post about your gene synthesis needs. I would suggest you check out Biomatik (www.biomatik.com). We are located in Ontario, Canada, and have extensive experience in gene synthesis. We are proud of our over 98% success rate with "No Gene, No Charge" policy.

Request a quote online and we will get back to you.

Best regards,

Theresa

A vector of I(1) variables yt is said to be cointegrated if there exist at vector βi such that βiyt is trend stationary. If there exist r such linearly independent vectors βi,i= 1,...,r, then yt is said to be cointegrated with cointegrating rank r.

I'm opting for AgeI+ SmaI double digestion.. but one generates cohesive end another blunt

Will that ligate in a same reaction with respective ends on my insert?

If your gene of interest is expressed via its own promoter (e.g. CMV, EF1a, etc) then you can always use the lentiviral plasmid in transient transfections, regardless of the lentiviral vector generation.

many thanks for answers, what about third rank tensor?

can we define a vector using some components of a third rank tensor?

Dear Stam and Peter, please pay attention that, we can simply obtain a vector from a third rank tensor using contraction of the third rank tensor. what do you think about this?

Hi,

I suggest you see the links on the topic.

Best regards

The Bloch vector of a qubit is a vector in three-dimensional space that represents the state of the qubit. It is defined as: ⃗ v = (vx, vy , vz ) where vx, vy , and vz are the components of the vector along the x, y, and z axes, respectively. Rotations of a qubit in the Bloch sphere can be represented by unitary matrices, and the same unitary matrices can be used to rotate the Bloch vector. Specifically, if we have a unitary matrix U that represents a rotation of the qubit state, we can apply this rotation to the Bloch vector by multiplying it by the matrix U †, where U † is the conjugate transpose of U . This is because the action of a unitary matrix on a quantum state is equivalent to a change of basis, and the conjugate transpose of a unitary matrix is its inverse, which reverses the change of basis. For example, suppose we have a qubit in the state |ψ⟩ = α|0⟩ + β|1⟩, where α and β are complex numbers such that |α|2 + |β|2 = 1. The corresponding Bloch vector is: ⃗ v = (Re(β), Im(β), Re(α)) Now suppose we want to rotate the qubit around the z-axis by an angle θ. The corresponding unitary matrix is: U = ( e )−iθ/2 0 0eiθ/2 The conjugate transpose of U is: U † = ( e )iθ/2 0 0e−iθ/2 To rotate the Bloch vector, we multiply it by U †, giving: ⃗ v ′ = U †⃗v = ( e )iθ/2 0 0e−iθ/2 ( ) Re(β) Im(β) Re(α) = ( e )iθ/2 Re(β) e−iθ/2Im(β) Re(α) This new vector represents the state of the qubit after the rotation around the z-axis.]Introduction Yes, we can rotate Bloch vectors for qubits just like we do with qubits in the Bloch sphere. 1

The Bloch vector of a qubit is a vector in three-dimensional space that represents the state of the qubit. It is defined as: ⃗ v = (vx, vy , vz ) where vx, vy , and vz are the components of the vector along the x, y, and z axes, respectively. Rotations of a qubit in the Bloch sphere can be represented by unitary matrices, and the same unitary matrices can be used to rotate the Bloch vector. Specifically, if we have a unitary matrix U that represents a rotation of the qubit state, we can apply this rotation to the Bloch vector by multiplying it by the matrix U †, where U † is the conjugate transpose of U . This is because the action of a unitary matrix on a quantum state is equivalent to a change of basis, and the conjugate transpose of a unitary matrix is its inverse, which reverses the change of basis. For example, suppose we have a qubit in the state |ψ⟩ = α|0⟩ + β|1⟩, where α and β are complex numbers such that |α|2 + |β|2 = 1. The corresponding Bloch vector is: ⃗ v = (Re(β), Im(β), Re(α)) Now suppose we want to rotate the qubit around the z-axis by an angle θ. The corresponding unitary matrix is: U = ( e )−iθ/2 0 0eiθ/2 The conjugate transpose of U is: U † = ( e )iθ/2 0 0e−iθ/2 To rotate the Bloch vector, we multiply it by U †, giving: ⃗ v ′ = U †⃗v = ( e )iθ/2 0 0e−iθ/2 ( ) Re(β) Im(β) Re(α) = ( e )iθ/2 Re(β) e−iθ/2Im(β) Re(α) This new vector represents the state of the qubit after the rotation around the z-axis.

Dear Tchorz. Maybe Dr. Ali Afgar could guide you.

To maximize the log-likelihood estimate of a signal using maximum likelihood estimation in MATLAB, you can use the built-in optimization functions. Here's a general process you can follow:

So it depends on the model you have at hand. Here's some papers applying MLE for different type of problems[1-3]:

[1] Bazzi, Ahmad, Dirk TM Slock, and Lisa Meilhac. "Efficient maximum likelihood joint estimation of angles and times of arrival of multiple paths." 2015 IEEE Globecom Workshops (GC Wkshps). IEEE, 2015.

[2] Bazzi, Ahmad, Dirk TM Slock, and Lisa Meilhac. "On a mutual coupling agnostic maximum likelihood angle of arrival estimator by alternating projection." 2016 IEEE Global Conference on Signal and Information Processing (GlobalSIP). IEEE, 2016.

[3] Bazzi, Ahmad, Dirk TM Slock, and Lisa Meilhac. "On Maximum Likelihood Angle of Arrival Estimation Using Orthogonal Projections." 2018 IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP). IEEE, 2018.

Feeling is: to present a matrix as an identity matrix multiplied by the vector M+xy Since the vector M+xy can be presented in the form of a matrix so it's product and the identity matrix will be the matrix which is equal to the original vector M+xy This one can be presented as lambda_1,... \lambda_{k-1}, \lambda_k+yx, \lambda_{k+1},... ,\lambda_n. If you take lambda outside of the brackets you will have (k+yx...n+yx) If majorization holds for a vector it will also hold for a vector times identity matrix as matrix is only a number of elements arranged in columns and rows But I can be wrong on this Better to say something than nothing So Im saying

There are two possible reasons why you can get self ligation with a double digested plasmid. First is that the stuffer fragment might act as insert in the ligation reaction giving you back the original plasmid. The second is that some fraction (even a tiny fraction) of the plasmid might not have been cut with one of the two enzymes, so this tiny fraction can readily recircularize itself. These are why the vector only religation is an important control to know the background in a reaction.

Thank You !! Shouxing Qu Aparna Sathya Murthy !!

Umbrella Sampling is a simulation method used in molecular dynamics to calculate the potential of mean force (PMF) along a reaction coordinate of interest. This PMF can be used to calculate thermodynamic quantities such as free energy and enthalpy.

The calculation of imaginary frequencies and displacement vectors from Umbrella Sampling requires a series of steps, including the following:

Preparation of the simulation: Umbrella Sampling requires the definition of windows along the reaction coordinate and the application of a biasing potential to each window to sample the conformations of the system.

Calculation of the PMF: The PMF can be calculated from the distribution of configurations in each window by using the weighted histogram analysis method (WHAM) or other similar methods.

Normal mode analysis: The PMF can be used as a potential energy surface (PES) for a normal mode analysis, which involves the calculation of the eigenvalues and eigenvectors of the system's Hessian matrix. The eigenvalues represent the frequencies of the normal modes, and the eigenvectors represent the displacement vectors.

Imaginary frequency analysis: The normal modes with negative frequencies are associated with unstable conformations and correspond to imaginary frequencies. The magnitude of these frequencies can be used to estimate the rate of conformational changes, such as conformational transitions or protein folding/unfolding.

Displacement vector analysis: The displacement vectors can be used to visualize the normal modes and the direction of the conformational changes associated with each normal mode. This can provide insight into the mechanism of the reaction and the key residues involved in the transition.

It's worth noting that Umbrella Sampling and the subsequent analysis can be computationally intensive, and it is essential to choose the appropriate parameters and simulation conditions to ensure accurate results.

Hello!

I have been doing very similar cloning for the past months. I have a 9kb insert separated into three fragments with 20bp overlapping regions to perform Gibson assembly.

If you are happy with how you designed the overlapping regions and primers and it works on Snapgene then I can give you the advice that worked for me when it comes to the actual cloning.

First, I'm guessing you are amplifying your three fragments by PCR, do you run them on a gel and do a gel extraction or do you just do a PCR clean-up straight away? If you are just doing a PCR clean-up with no gel extraction then a lot of non-specific amplicons could also be purified along with the actual 3kb amplicon. These non-specific amplicons contain the homology region that Gibson will use (because the primers used to amplify them have the homology region as well), and because they are shorter they will be incorporated into your vector more efficiently than your actual 3kb fragment. If you haven't done it yet, run a gel with your PCR product without PCR purifying it and see if there are any non-specific bands, if there are non-specific bands then run all your PCR samples on a gel (without previously purifying them) and gel extract the appropriate band. If your PCR is super neat and there are no non-specific bands then this might not be necessary but I would still recommend it.

The problem with doing a gel extraction instead of a PCR purification is the high amount of impurities that the extracted DNA has (salts, etc.) these will inhibit the Gibson reaction. To fix this you can do a PCR purification on the gel-extracted DNA. Although you will lose DNA quantity, not necessarily DNA concentration if you take care to dilute it in less water. This step is critical.

You can also digest the backbone overnight or for longer periods and also run it on a gel for longer to make sure you reduce the number of undigested contaminants as much as you can when extracting it. Remember to also PCR purify your gel-extracted backbone!!

When doing the Gibson assembly, you can run it for 4 hours instead of 15 minutes which can increase efficiency. I used Gibsons assembly from NEB, then changed to their HiFi assembly mix which is essentially the same but better, and it worked for me.

Hope you manage to clone it!

Yes PDONR221 can be used as entry vector entry gateway system

pDONR221 (12536-017): empty middle entry vector for generation of new middle clones.

empty 5' and 3' donor vectors for generation of new 5' and 3' entry clones. Note: the catalog number provided here is for the MultiSite Gateway Three-Fragment Vector Construction Kit; it includes pDONR221 as well as a destination vector (pDest R4-R3). This appears to be the only way to purchase the empty donor vectors at this point.

The Gateway cloning method, invented and commercialized by Invitrogen since the late 1990s, is the cloning method of the integration and excision recombination reactions that take place when bacteriophage lambda infects bacteria. This technology provides a fast and highly efficient way to transport DNA sequences into multi-vector systems for functional analysis and protein expression using Gateway at sites.

What does the no-ligase vs + ligase control look like? If you have same number of colonies without ligation, then you have background of uncut vector. If you only get colonies upon ligation then either you have some vector only singly digested and not double digested or else your insert is not so good so the frequency of ligation products is low. Did you screen a large number of colonies before saying you had no inserts?

Regarding digestion: presence of 250 bp band suggests, that at least part of your plasmid has been cleaved. But this is never 100% effective and at least small fraction of uncleaved vector will markedly affect your result. Most of your clones will be empty.

Thank you for your answer Aparna Sathya Murthy