Science topics: Vectorization
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Hello everyone! I will summarize my situation: I am performing KO using CRISPR/Cas9 for PTEN in Prostate Cancer cells (DU145 and 22RV1). I am using a commercial vector that includes the guides, Cas9, puromycin resistance and GFP. I have infected the cells with the virions carrying the guides, I have done the selection with puromycin and I have even done a first western blot of PTEN. In this Western of the cell pool I have seen a large decrease of PTEN with respect to the Wildtype cells, which means that it has worked to some extent and now I must isolate clones to be able to obtain a complete KO. However, when I have gone to look at GFP expression on the fluorescence microscope I have not seen any of the green cells (I have also used GFP expressing cells as a positive control to make sure there is not a glitch with the microscope). I don't quite understand why you see a decrease of PTEN in the WesternBlot but I am not able to see GFP if in theory they go in the same vector (The vector is Merck's LVL01). Can anyone know what is going on? is there any explanation? should I start again from the beginning? Thank you very much in advance
I'm still an amateur and don't know where researchers usually get their research materials for biological research. I would like to ask where to obtain the lentivirus vector carrying the OSKM factor for reprogramming
I am trying to figure out how to make more TOPO vector. I'm using the PCR4Blunt-TOPO vector which is already linearized. In the kit there are only 25 ul of 10 ng/ul TOPO vector and I have do to several transformations using this vector. I was therefore wondering if it is possible to make more TOPO vector by doing transformation just without adding any insert. So basically I want the TOPO vector to self-ligate. The vector has blunt ends and as far as I understand, there are phosphates missing on the ends of the backbone, making it harder to self-circularize. Any suggestions on how this could be done?
Hello All,
I'm trying to clone a 1.5 kb fungal gene to a PJET cloning vector (Thermo scientific) that has 98% selection capability, and I'm also verifying the clone using the PCR, but every time after sequencing, I get the result as a vector sequence rather than the specific clone fragment that I'm targeting. Please suggest me if any one aware about this problem?
I want to clone my 60 bp insert into my approximately 7000 bp vector. I did the ligation with a vector to insert ratio of 1:10. Either very few or no colonies grew. Also my colony PCR results were negative. How should I change the vector insert ratio?
Hello everyone,
I designed primers for Gateway cloning, but I'm unsure about the sequences I need to add. I plan to transfer my genes along with their native promoters into a donor vector. I added GGGG-attB1 and 2 seq-gene to forward and reverse primers. According to the information I gathered, I need to make changes to the stop codon region related to the destination vector, which I will use for the LR reaction. My destination vector contains a GFP tag. In this situation, should I design my reverse primer starting from the stop codon-containing region and eliminate the stop codon? If you have any additional insights that might be important for this experiment, could you please share them?
Thank you...
I have been struggling with getting clones with LR plus reaaction to clone three fragments into a destination vector. I use 5fmol of entry clones and 10fmol of the destination vector. I first calculate the fmol : (concentraion in ng x 10^6)/ (size in bp x660). I then calculate and dilute the sample and keep the reaction. for eg: if i get 29.9 fmol in concentration for an entry clone and to bring it to 5fmol, i would take 1.67uL of the plasmid and dilute in 8.33uL water and from it take 1uL for the reaction. So, I am taking 1uL of all three entry clones (of 5fmol each), 1uL of destination vector (10fmol) and 1uL of LR plus clonase. Can anyone suggest what I am doing wrong?
Hi there!
I am trying to clone with the LR Clonase enzyme (LR Clonase II, Thermo Fisher). I have an entry vector for each gene of interest (genes with a length of 120 and 490 bp approximately), which I am trying to recombine with the destiny vector (Gateway pK2GW7). I performed the recombination as indicated in the protocol and then transformed E. coli DH5 alpha bacteria but I am unable to obtain colonies with the constructs. The enzyme works
Things I tried:
- Check the sequences of the entry vectors: they are correct and checked by sequencing.
- Linearize the entry vectors
- Use 150 ng of destiny vector and 10 ng of entry vector for recombination.
- Leave recombination over night
- Cut a fragment of the entry vector to linearize it because it recirculates and has the same antibiotic resistance as the destiny vector. The few colonies I got after transforming were with the entry vector.
I don't know what else to try to get the clones. What do you recommend? Do you think the size of my genes affects the recombination in any way?
Thanks to all!
I'm trying to perform ligation by cleaning up the insert and vector prior to ligating them. Concentration is coming down to very low after the cleanup which is not giving good results. Pls help me .
Hello everyone,
I am working on a project focused on digitizing and vectorizing archival maps and forest inventory sheets. The goal is to integrate these documents into a spatial database to analyze forest dynamics influenced by fires, logging, and climate change.
I am looking for ways to automate the digitization and vectorization processes, particularly using shape recognition, image segmentation, and machine learning tools to convert map features into vector polygons.
If you have recommendations for effective tools or methodologies for these types of documents, I would greatly appreciate your insights. I will also be sharing some visual examples to illustrate these maps and sheets.
Thank you in advance for your suggestions!

Hello, I performed an in vitro transcription of a PCR product cloned into the PCR 4.1 vector. I used the T7 enzyme for the transcription. I need this transcript to be used as a control in quantitative RT-PCR. How can I ensure that the T7 enzyme transcribes only the cloned insert and not beyond, considering that the vector does not have a specific termination site for the enzyme?
I have been doing gibson assembly for months, amplifying vector and inserts separately, then doing the assembly reaction at 50 degrees for an hour following all the instructions using 100 ng vector DNA and abundant inserts (as planned to insert one gene for the vector). I screen the transformed colonies by plasmid purification, and I got some positive results with the amplification length, same for the gene length, but when I send it for sequencing results, all come negative with blank vector. I tried rechecking the result by single RE digestion, but the result is abrupt (the linear DNA is not aligning with its own length along with the NEB marker). What will you be that I will be checking? any suggestions? Where does the problem lie?
#cloning #Gibsonassembly
In the first figure (empty vector), only a short linker is in front of EGFP. EGFP is followed by IRES2 and mCherry. The cells transfected with this empty vector only express mCherry; I can not get any EGFP-positive cells via flow cytometry, which means EGFP was not expressed. Theoretically, the EGFP: Cherry ratio should be equal to 1.
However, in the second figure, I inserted the sequence of POI in front of the short linker and EGFP. This time, EGFP can be expressed.
So I am wondering if that is something wrong with my empty vector that leads to EGFP not being expressed.


This discussion concerns the positivist versus the realist interpretation of quantum non-locality in the framework of EPRB experiment. It's about the possibility to change this question of interpretation into a falsifiable proposal: the conservation (or not) of 2-time correlations on Bob's side as long as only Alice performs polarization measurements.
More precisely, the article "Each moment of time a new universe" (https://arxiv.org/abs/1305.1615) by Aharonov, Popescu and Tollaksen, presents:
- a T-symmetric formulation of the temporal “evolution” of a quantum system which does not evolve (H=0)
- a very important consequence predicted thanks to this formulation concerning the interpretation of the EPRB experiment.
Cf. this very interesting 8 pages article (https://arxiv.org/pdf/1305.1615) and a video presented by Popescu (https://www.youtube.com/watch?v=V3pnZAacLwg).
Thanks to their 2-state vector T-symmetric formalism (https://arxiv.org/abs/quant-ph/0105101), Aharonov, Popescu and Tollaksen notably highlight the following facts:
- as long as no quantum measurement is carried out on a given quantum system (undergoing a H=0 Hamiltonian evolution) the 2-time measurement O(t2) - O(t1) between instants t1 and t2 vanishes whatever the observable O. This proves the existence of a time correlation between successive states of a quantum system as long as it doesn't undergo any quantum measurement.
- On the contrary, the correlation O(t2)-O(t1) = 0 is broken between instants t1 and t2 respectively preceding and following a quantum measurement (except in the specific cases when the measurement result is an eigenstate of O).
Concerning EPRB type experiment, this document indicates §Measurements on EPR state:
- The break, on Alice's side, of the 2-time correlations between instants t1 and t2 preceding and following a quantum measurement by Alice. Indeed, except in a particular case when the measurement result is an eigenvalue of O, the 2-time correlation O(t2) - O(t1) = 0 is lost.
- The conservation, on Bob's side, of the 2-time correlations O(t2) - O(t1) = 0 as long as Bob doesn't make any measurements on his side.
Thus, the 2-state vector time-symmetric formalism shows the asymmetry of the quantum state obtained, during an EPRB experiment, after a measurement carried out on one side only. That asymmetry doesn't show up in the standard formulation. Consequently, the standard one-state vector time-asymmetric quantum formalism suggests a (hidden) relativistic causality violation. On the contrary, the conservation of the 2-time correlation in the 2-state vector formalism provides, in my view, a proof that, on Bob's side, nothing happens as long as only Alice carries out quantum measurements on her side.
This seems providing a testable prediction allowing us to decide between:
- a realist interpretation of the EPRB experiment where the quantum state is interpreted as the model of an objective physical state (cf. On the reality of the quantum state, https://arxiv.org/abs/1111.3328) and the reduction of the wave packet as instantaneous, non-local AND objective, cf.:
- Bohm, Bell https://web.archive.org/web/20190104202702/http:/www.tcm.phy.cam.ac.uk/~mdt26/PWT/lectures/bohm5.pdf
- Goldstein https://arxiv.org/abs/0903.2601
- Valentini https://arxiv.org/abs/quant-ph/0112151
- Percival https://arxiv.org/abs/quant-ph/9803044
- Hemmick https://arxiv.org/pdf/quant-ph/0412011
- Special Relativity and possible Lorentz violations consistently coexist in Aristotle space-time https://arxiv.org/abs/0805.2417 ...
- on the contrary, a positivist interpretation of the EPRB experiment, the instantaneous and "non-local reduction of the wave packet" is interpreted as an irreversible and local record of information, hence up to be read by an observer carrying out the measurement, without objective change of Bob's photons state when only Alice performs polarization measurements on her photons. cf.:
- Rovelli https://arxiv.org/abs/quant-ph/0604064
- Jaynes https://bayes.wustl.edu/etj/articles/cmystery.pdf (1)...
When only Alice carries out measurements on her side, the prediction of the conservation of the 2-time correlation on Bob's side, resulting from the 2-state vector time-symmetric formalism, decides, in my view, in favor of the positivist interpretation of the EPR non-locality. In my view, the positivist interpretation becomes a falsifiable physical postulate instead of a pure philosophical question.
Such an experimental verification seems solving a 40 years debate between positivist and realist interpretations of Bell's inequalities violation. Hence, this experimental validation seems deserving to be carried out (but I don't know if it has already been Achieved).
Would you agree with this view?
(1) Note, however, that E.T. Jaynes supports a realist interpretation of physics and its role despite, paradoxically, his insistence on the importance of Bayesian inference and the broad development he gave to this approach (cf. Maxent https://bayes.wustl.edu/etj/articles/rational.pdf)
I have been trying to clone a genomic region (Promoter + InEx) that sums up close to 10kb, so far it has been unsuccessful with restriction based cloning and have opted for a gateway strategy which now too has become a headache. I am amplifying my fragment with attB1 and attB2 sites added to my primers and afterwards proceed to purifying from the gel. I have also done the respective calculations to leave the reaction equimolar with the pDONR201 plasmid and left incubating the reaction overnight (up to 18 hours). Afterwards I transform DH10B E. coli and end up with 1 or 2 colonies growing on kanamycin plates if any at all and only after a long incubation!!!! I have also tried a Carbenicillin resistant version of pDONR221 and have only gotten empty vectors which in principle should not happen since there is also de presence of ccdB within the vector?? I have also tested different BP Clonase II batches and have ruled out the enzyme mix.
If anyone has any experience in cloning fragments this size or has had to deal with any of these issues before I would appreciate any input!
I'm experiencing persistent issues with cloning three genes (1.1 kb, 1.2 kb, and 2.1 kb) into a 5.4 kb vector. Despite multiple attempts using the same conditions, I consistently fail to get colonies on the transformation plates. My positive control (undigested plasmid) works fine, and the negative control (restricted plasmid) shows no colonies, suggesting that my transformation and restriction digestion are working correctly.
However, I’ve been struggling with ligation, even though in one of my attempts last week I managed to get a single colony, which was confirmed positive. Although the insert band (1.2 kb) from purified plasmid after restriction digestion was faint, the PCR amplification from the colony gave the expected size, leading me to assume the colony was positive. Since then, using the same controls and protocol, I've tried cloning the other two genes but have not obtained any colonies. Here is the detail of the steps I am following along with the controls.
Step-by-Step Procedure
1- PCR Amplification
The PCR amplification is working well, with clear bands of the expected sizes.
2- Gel Extraction
I'm using the GeneJET Gel Extraction Kit to purify the PCR products after electrophoresis. The elution buffer is warmed, and I elute the product from the gel to 20 μL buffer.
3- Restriction Digestion
I'm using Thermo Fisher Fast Digest NdeI and BamHI enzymes for the restriction digestion. As a control I am doing restriction digestion of a recombinant plasmid using the same enzymes and getting two bands for vector and insert, confirming the digestion is successful.
4- Purification and gel analysis
After restriction digestion I purify the restricted genes and vector form gel using the same GeneJET GEL extraction kit with warm elution buffer and elute the 50 μL restriction reaction to about 20 μL of elution buffer after cleaning the product I run about 4 – 5 μL of the cleaned products on gel to check that I have the genes and vector to ligate.
5- Ligation
For ligation, I’m using a 1:1 or 1:3 vector-to-insert ratio with the following reaction setup:
Water: 10 μL
Vector (50 ng): 3 μL
Insert (40 ng): 4 μL
Ligase buffer: 2 μL
T4 DNA ligase (1 μL)
The ligation reaction is carried out at 16°C for 3 hours, followed by 22°C for 30 minutes and a 10-minute inactivation at 80°C. I have also tried ligation at 16 oC for 30 minutes, 1 hour, 2 hours, and overnight. The recommended ligation time with my enzyme is 15 to 30 minutes.
6- Transformation
In my transformation trials, I’ve used 2 μL, 2.5 μL, 4 μL, and 5 μL of the ligation reaction but haven’t obtained any colonies, except for one faint band from the single colony I previously isolated. For control, I am taking 1 μL of the plasmid as positive control and the same amount of the restricted plasmid as negative control as the ligation mixture. I always get colonies on the positive plate which means the transformation procedure is fine.
7- Gel Analysis of Ligation
The ligation reaction often shows multiple higher molecular weight bands on the gel with vector and insert bands that are sometimes too faint and sometimes only the vector band is visible along with high molecular weight bands.
Dear all,
Can anyone tell me how to fit nonlinear mixed models by the first-order (FO) and first-order conditional expectation (FOCE) methods ? I only know that a Package called nlme can be used to fit nonlinear mixed models by nlme(model, data, fixed, random, groups, start, correlation, weights, subset, method, na.action, naPattern, control, verbose) in R software.
But I don't know which method (FO or FOCE) was used in nlme exactly. Both FO and FOCE methods linearize a nonlinear mixed model through a first-order Taylor series expansion.They differ on how random parameter vector bi is predicted and how subsequent SS predictions are generated. I got that Nonlinear mixed models can be fitted by the SAS macro NLINMIX by incorporating random parameters into these two models.But how to do it in R software? And I upload an article about the question. Can you help me? Thank you very much!
HoPui
I feel puzzled that HIV mainly infects immune cells because it needs CD4 as receptor and CCR5/CXCR4 as co-receptors for entry, but when some toxic genes are deleted and geome taken apart for vector use, it works efficiently in a wide range of cell types(for example, MCF-7 and HEK293FT).
I'm using solar load model in FLUENT, where the sun vector direction is set to (0, 0, 1) (sun rays are incident vertically down from the top wall), but I don't know why the sun rays are always tilted (instead of vertically down) inside my model.

Hi, I am making a 3rd generation LV expressing my gene of interest. I know that putting a polyA signal within the LV genome will lower the vector titer, but what happens if I put it in the opposite direction? Did anyone try?
Thank you for reading my question.
Are newer RFPs like mRuby3 or mScarlet better complements for eGFP? Or should I stick to the eGFP/mCherry pair?
I am new to FRET and I am planning a FRET experiment to study interactions between a mutated soluble mammalian protein (which forms aggregation) and a wild type protein in cytosol.
My cells are HEK293T transferred with the two strains of plasmids. Confocal Laser Scanning Microscopy will be applied to conduct a short live-cell imaging. The microscope is Zeiss LSM880.
Feasible emitter wavelengths are: 405 nm, 488 nm, 543 nm, 594 nm, and 633 nm.
Both vectors carrying a fusion protein of object-eGFP have been already constructed and validated in previous experiments.
After conducting some literature review, it seems that CFP/YFP pairs and GFP/RFP pairs meet my needs. Considering that I have object-eGFP vectors and the blue emitter (488nm) is not working, I prefer a GFP/YFP pair.
Effective pairs like mNeonGreen/mScarlet-I and mClover/mRuby3 have been validated.
To the best of my knowledge, The pair eGFP/mCherry has been well-established and reliable. However, relatively low Quantum Yield and Extinction Coefficient of mCherry still raise my concern.
Thank you in advance for your reply!
I am using the Gibson HiFi Assembly Kit to assemble a DNA fragment. Both the DNA fragment and the vector are approximately 1000 bp in length. Note- I am not performing a PCR step. After completing the Gibson assembly reaction, I can observe the desired band corresponding to the insert. However, after performing E. coli transformation, selecting specific colonies, extracting plasmids from them, and running them on a gel, I observe bands that are not of the desired length.
What steps should I take to ensure I obtain the correct bands or vector size of the desired length?
I was able to transform bacteria sucessfully with small inserts (+-500bp and 1500bp) using infusion technic. However, when it comes to larger inserts (5500bp and 6000bp), it doesnt work. We already follow the troubleshooting guide descript on the protocol, and tried differentes approaches (concentration, proportion, longer incubation).
Our primers were designed following Takara instruction with 15bp of homology and were already checked.
Our linnearized plasmid was diggested by Xho1 and Sal1 and it its 5004bp long. The final concentration of the linnearized plasmid is 195ng/uL. Our insert is 5542bp (larger than the vector) and its final concentration after purification is 27ng/uL. I'm using competent E. coli Stbl3. We use the concentration around 50ng/uL up to 150ng/uL in the infusion solution.
We tried to transform bacteria by using different proportions between the vector and the insert (1:1; 1:2 and 1:3 each). We also incubated the infusion solution for 1 hour at 50ºc (even knowing that the protocol says longer is no better). I already checked the reagents by using the positive control.
We use the heat-shock protocol, by defreezing bacteria for 30 minutes in ice; adding the infusion solution (3uL) on bacteria and leting it incubate for 30 minutes in ice; then we heat shock the bacteria for 45s at 42ºc and quickly put them into the ice again. Final step, we plate it in a petri dish with agar LB and streptomycin and let it incubate for 16-20h.
The thing is that we dont have any colony and when it appears, it doesnt have our interested insert. I dont know what else i can do.
Dear all, according to the equation, we can get the inverse relaxation time for the ionized impurity scattering mechanisms. But I don't know how to get the epsilon and electron wave vector. Looking forward to your answer.

Hi everyone,
I wanna clone a 96bp insert fragment in a vector. I simulated in Snapgene, but 5' end of my reverse nucleotide is vice versa (It is TTAA instead of AATT).
Is it possible to change 4nucleotides by PCR instead of synthetizing the whole oligonucleotide?
Share your thoughts and procedure of waste disposal.
since the siRNA is not loaded onto a vector and is supposed to be transfected alone using lipofectamin 3000,the process is more difficult and requires more troubleshooting.I would greatly appreciate it if anyone with experience could provide tips or strategies to improve the transfection efficiency.
thank you in advance!
CO2 Sequestration
1. To what extent,
the concept of ‘impelling force’
introduced by Hubberts
would be able to provide
a useful means of
visualizing
the net forces acting on CO2?
2. If the impelling force represents
the negative of the gradient in CO2/brine potential,
will it still remain to be a vector quantity
that would precisely define
the direction
in which
CO2 would tend to migrate,
considering
capillary effects?
Suresh Kumar Govindarajan
Good morning everyone
I have created the pseudomonas oryzihabitans mutant via Gibson assembly (homologous recombination) with the suicidal vector pK18mobsacB . And now I am trying for complementation of the gene with the same vector i.e pKmobsacB but I am not able to transform the plasmid in the pseudomonas mutant. Please suggest what could be the possible reasons for transformation failure or there is a problem with the vector as I am using same vector for deletion and complementation? As I searched many papers but no one have gone for gene deletion and complementation with the same vector.
Hi,
I am having troubles cloning a Cas9 gene (around 4300 kb) into a vector, replacing a LacZ cassette. (I use the uloop system.) I use SapI as a restriction enzyme and T4 as a ligase. I cloned several other constructs into the same kind of vector with high efficiency (mainly white colonies, and almost all carry the correct insert when screened).
When I try to insert Cas9 (or also dCas9) I yield the same number of colonies, with again almost all white. However, when I screen the colonies via colony PCR or restriction digest the isolated plasmids of the colonies, no vector carries the insert. I did not sequence these plasmids, but from the restriction digest it suggest, that the plasmid only lost its LacZ cassette and was closed again without any insert (eventhough overhangs are not compatible).
I tried two settings for GGC:
37 C 5min...............37 C 5min
16 C 5min................16 C 5min
(25cycles)................(40cycles)
65 20min-................65 20min
85 10min..................85 10min
4 hold......................4 hold
I tried two different buffer conditions:
-T4 buffer only
-Or 50% T4 buffer, 50% Cutsmart buffer
I started with fresh Cas9 (and dCas9) PCR templates and with several fresh receiver plasmids. Also I used different competent cell batches.
I double checked the overhangs of receiver plasmids and the overhangs created on the Cas9 insert.
I am running out of ideas..
Does anyone know if GGC with single large inserts (4000 kb+) effect GGC efficiency?
Does anyone have a suggestion how I could improve GGC efficiency?
Further troubleshooting suggestions?
I really would appreciate your ideas!
Thanks,
Florian
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.
I was trying to clone an RCA product (digested with a single restriction endonuclease) into the pUC 19 vector but I failed. After cloning, I have to go for the sequencing. Can I clone it into a TA vector?
Vectors which can accept PCR products up 700-800bp
We utilize the pET22b vector for cloning and aim to incorporate the pelB signal sequence for periplasmic expression. For this purpose, we are using the restriction enzymes NcoI and XhoI. However, we have encountered a frameshift during translation due to NcoI. Does anyone have suggestions on how to resolve this issue?
Hello
I am using two expression vectors (pCAGGS and pCDNA) for transforming a fragment of gene (2kbp). For transformation, I am using NEB 10 beta-competent E.coli cells.
Issue?
Although my gene of interest is transformed into the expression vector, a portion of the expression vector's nucleotide sequence is being removed or deleted.
Could anyone share their experience on this and how you overcame this problem?
Thanks in advance!
I want to demonstrate the vector transmission-line equations (2.16) and (2.17) from Sergei Tretyakov's book "Analytical modeling in applied electromagnetics" pages 18-19. Any help please.
I want to insert a gene in a vector using restriction cloning, but the enzyme that I have to use has three restriction sites in the vector. It is imperative that I use only this enzyme and no other, so I can't use a different restriction site or other enzyme. Can someone help with this issue?
I have tried partial restriction digestion with different units of enzyme as well as different time periods of incubation but haven't got a single band. The enzyme is cutting at all three sites whatever conditions I try in the partial restriction digestion.
I am cloning a gene (903bp) into miniTurbo_NLS (6338 bp).
- I PCR amplified the insert from it's backbone to introduce EcoR1 and Xho1 sites. It was run on gel and eluted. I then double digested it for 5 hrs at 37oC and was eluted from gel.
- I double digested the vector for 5 hrs at 37oC and did alkaline phosphatase treatment and inactivated. I then ran it on gel giving a band of 6248 bp and eluted. Note- I didn't confirm the 90 bp fallout.
- The vector and insert was ligated in 1:3 ratio but no colonies grew after transformation into TG1 chemically comp cells.
- Ligation was attempted again with 1:7 ratio and only 1 colony grew.
- The control plate with vector only + ligase had 3 colonies.
- Another control plate with vector only - ligase had no growth.
- The single colony that grew was insert specific colony pcr negative.
- Even after isolating the plasmid it was negative.
How to make this cloning work ?
Hello everyone,
I am currently running a luciferase assay using the Dual-Glo® Luciferase Assay System (Promega) to assess miRNA-MRE interactions. I cloned a combination of three MREs downstream of the Renilla translational stop codon in the psicheck2 vector. For the experiment, I am co-transfecting psicheck2_MRE with pcDNA3_mCherry plasmids containing a miRNA specific for the MRE. My experimental conditions are:
- psicheck2_MRE + pcDNA3_mCherry_miR1_2
- psicheck2_empty + pcDNA3_mCherry_miR1_2
- psicheck2_MRE + pcDNA3_mCherry_empty
- psicheck2_empty + pcDNA3_mCherry_empty
- Non-transfected cells
My expectation is that the miRNA will bind to the MRE and reduce Renilla luciferase activity in the psicheck2_MRE + miRNA condition. While I am observing this reduction as expected, I am also seeing a decrease in luminescence in psicheck2_empty in the presence of the miRNA, which is unexpected.
Additionally, the Renilla levels of the empty vector are considerably lower than the vector containing the MRE. I normalized to the empty vector and then to the no miRNA control, but I’m finding it difficult to trust the results due to the unexpected impact on the empty vector.
Has anyone experienced similar issues? How would you suggest troubleshooting or interpreting the effect of miRNA on the empty vector?
Any insights or advice would be greatly appreciated!

Hi everyone,
I'm interested in analyzing exchange rate behavior by testing the Dornbusch model of overshooting exchange rates, however I still struggle with finding the correct mehtodology. How should I proceed? I think by applying time series analysis components such as Vector moving average or similar instruments this would be possible - what do you guys think?
Kind regards
Zan Blagojevic
I am conducting a transformation using a 300 bp insert and an empty vector of approximately 300 bp. After successful transformation and colony growth, I performed colony PCR. However, the gel consistently shows a 300 bp band, which suggests that the colonies contain the empty vector without the insert. Given that I expect a 600 bp product (300 bp insert + 300 bp vector), what might be causing this issue? (Cloning technique: LIC).

This is a simple proof the guitar is Hamiltonian. Then by deconstruction so is string vibration because the string is the smallest open set on guitar.
The time-independent Hamiltonian has the form H(p, q) = c and dH/dt = 0.
All I need is to define p and q.
So p will be the center of harmonic motion, and q will be a potential energy gradient that reads off the differential between any two points.
Consider the set of notes for the guitar tuning known as standard: E A D G B E.
The tuning naturally separates into two vectors in this way: Indexing the tuning notes by counting up from the low E the pitch values are equivalent to p: 0 5 10 15 19 24.
Now taking the intervals between the notes we have a second vector q: 0 5 5 5 5 4.
It is important to notice that tuning vectors p and q are equal, opposite, and inverse, which is expected since the orbit and center have this relation in the Hamiltonian.
For instance, p is the summation of q and q is the differential of p. The points in p and the intervals in q together make a unit interval in R.
Most important, p = 1/q means the tuning is the identity of the guitar. If you know the tuning, you know everything (all movement). You can learn guitar without learning anything but the tuning.
The proof the vectors are Hamiltonian is this, p is the center of motion in R6, and q is the gradient of the potential field surface in R5 where every vibrational state is presented by a single point.
The coordinates of notes on guitar chord charts given by the gradient function
form a union as a smooth atlas.
Therefore, it must be true the guitar is Hamiltonian. How else could the symplectic manifold be smooth?
Physicists and mathematicians have no choice but to accept that one degree of freedom is better than two. The fact that they cannot see it implies an illness of the public mind that cannot think straight about classical mechanics.
I have to do cloning gene in pseudomonas, but we didn't a suitable vector. Does anyone know how can I design pucp18 from puc18 vector? Just, I add replication origin of pseudomonas, can I clone genes? and how can I do that?
I designed what I thought would be a straightforward piggybac (https://en.vectorbuilder.com/resources/vector-system/pPB_Exp.html) vector with the IL2-6xHis ORF downstream of an E1Fa promoter. The vector was transfected into HEK293 cells. The cells underwent selection for several generations and exhibited nearly 100% blue fluorescence (my marker contained BFP). I felt very safe that there would be strong expression of the vector but was disappointed to find no notable protein band corresponding to the size of IL2, both via coumassie and silverstain. I also used a His tag detection strip, but there was no hint of expressed IL2 protein... Any thoughts on what could have gone wrong? Anyone also trying to express cytokines using HEK293 cells?
why am i getting to see growth of yeast colonies after performing dilution spotting on the respective dropout media when only the empty prey and bait vectors were co-transformed? the yeast strain used was AH109 and the vectors used are PGADC1 and PGBDUC1
I've formulated a new foundation for physics, based on the discovery of the quantum circulation constant k, with a value equal to c*c but a unit of measurement in [m^2/s], with which we can define the time derivative of any given vector field [F] in physical three dimensional space time as follows:
d[F]/dt = -k Delta [F],
with Delta the vector Laplacian, THE second spatial derivative in three dimensions, which would be d^2/dx^2 in 1D.
Quite frankly, this is the equation that will one day be recognized as one of the biggest scientific breakthroughs of the 21st century, because there is simply no argument to be made against such a simple and straightforward application of the vector LaPlacian.
And this equation allows us to define higher order LaPlace and Poission equations, like for the velocity field [v]:
[a] = d[v]/dt = -k Delta [v],
[j] = d[a]/dt = -k Delta [a].
This in contrast to what has been done heretofore, namely using the grad, div and curl operators to define fields (Maxwell, Navier-Stokes), but no one managed to work directly with the vector Laplacian Delta to tie all things together. And whereas both Maxwell as Navier-Stokes are incomplete first order models, we can now formulate a second order model using higher order math.
One of the results of that is that the rather complex wave equation,
( Delta - 1/c^2 d^2/dt^2 ) [v](r,t) = 0
can be simplified to:
[j]/c^2 + [a]/k = 0,
illustrating the expressive power of this math and showing that we do need a second order model in order to describe space-time dynamics properly and completely.
Read all about it in my (very preliminary) notebook:
ChatGPT:
"In summary, while Maxwell’s equations provide a mathematically valid formulation, the new model offers a more physically consistent framework by rigorously separating linear and angular components, avoiding the blending of different types of behavior and ensuring adherence to fundamental principles of vector calculus."

My question concerns the QiagenTM Spin Miniprep kit and the purification of the Low copy pACYC vector from the E.coli DH10b genetic background.
When trying to purify pACYC vectors from the E.coli DH10b genetic background, I have repeatedly noticed that my cell pellet isn't lysed but redissolved after the addition of buffers P2 and P3 to buffer P1. The expected floating cloud of cell debris isn't generated and the lyseblue reagent produces a strawberry yoghurt like color instead of the expected indigo blue color. Surprisingly, this problem only occurs when using the DH10b nature.
Has anyone ever encountered this problem?
Thanks in advance!
Based on general relativity, gravity causes the curvature in space and time. In comparison to the structure of an electric field, what would be the field vector shape and structure of the gravity field? how it affects far-distance objects.
In computation of surface gravity for BTZ black hole, I cannot get the same result in literature, e.g. Eq(1.15) in gr-qc/9506079, even I cannot get the result of null surface, i.e. $\xi_a\xi^a=0$ for horizon Killing vector $\xi_a$(see Eq(1.13) in gr-qc/9506079). Could the results in gr-qc/9506079 be incorrect?
I am working on QCA designer tool. As soon as I click on "simulation type setup" the tool closes abruptly. I am unable to set a vector table for simulation in this case. Pls help me regarding this.
Simplifying the dataset is a crucial step in managing large datasets effectively in QSWAT and QGIS. Here’s a detailed guide on how to simplify your dataset:
1. Reduce DEM Resolution
Why: A high-resolution DEM (Digital Elevation Model) provides detailed topography but can be resource-intensive. Reducing the resolution can help balance detail with performance.
How:
- Resample the DEM:Open QGIS and load your DEM. Go to the menu "Raster > Conversion > Translate (Convert Format)". In the dialogue box, click on the "..." button next to "Additional command-line arguments". Replace X and Y with the desired pixel size (e.g., 100 100 for a 100-meter resolution). Enter the following argument to resample the DEM: bashCopy code-tr X YSave the output file and click "Run".
Effect: This reduces the DEM's resolution, decreasing the number of cells, which speeds up processing without significant loss of overall watershed characteristics.
2. Clip the Dataset
Why: If your study area is a small portion of a large dataset, processing the entire dataset is unnecessary. Clipping reduces the area to only what's required.
How:
- Clip the DEM:In QGIS, load the DEM and the boundary shapefile of your study area. Go to "Raster > Extraction > Clip Raster by Mask Layer". Select your DEM as the input layer. Choose your boundary shapefile as the "Mask Layer". Check the option "Match the extent of the clipped raster to the mask layer". Save the clipped raster and click "Run".
- Clip Vector Layers (e.g., Land Use, Soil Data):Load your vector layers (e.g., land use, soil data) and the boundary shapefile. Go to "Vector > Geoprocessing Tools > Clip". Choose your vector layer as the "Input Layer" and your boundary as the "Overlay Layer". Save the output file and click "Run".
Effect: Clipping the dataset reduces its size, making it easier and faster to process.
3. Simplify Vector Layers
Why: Vector layers with high vertex density (e.g., detailed polygons) can slow down processing. Simplifying the geometry reduces the number of vertices.
How:
- Simplify Polygons:Go to "Vector > Geometry Tools > Simplify Geometries". Select the vector layer you want to simplify. Adjust the tolerance level (higher values remove more vertices). Save the output and click "Run".
Effect: Simplifying reduces the complexity of vector layers, improving processing speed without a significant loss of accuracy.
4. Use Raster Compression
Why: Large raster files (like DEMs or satellite images) can be compressed to reduce file size without losing much detail.
How:
- Compress Rasters:Go to "Raster > Conversion > Translate (Convert Format)". Under "Advanced Parameters", add: diffCopy code-co COMPRESS=LZWSave the output file with the new compression settings.
Effect: Compression reduces file size, making it faster to load and process, especially for large areas.
5. Aggregate Raster Data
Why: If detailed raster data is not necessary, aggregating it to a lower resolution can reduce processing time.
How:
- Aggregate Raster:Go to "Raster > Analysis > Aggregate". Choose the input layer (e.g., land use raster). Set the aggregation factor (e.g., 2x2 cells to one cell). Save the output and click "Run".
Effect: Aggregating reduces the number of raster cells, simplifying the dataset.
6. Remove Unnecessary Layers
Why: Having too many layers loaded in QGIS can slow down the software, especially when handling large datasets.
How:
- Unload Unused Layers:Review all layers currently loaded in QGIS and remove any that aren't necessary for your current task.
Effect: This reduces the memory load and improves QGIS’s performance.
7. Simplify Using External Tools
Why: Some tools outside of QGIS (like GDAL or Python scripts) might handle large datasets more efficiently for specific preprocessing tasks.
How:
- Use GDAL Commands:GDAL offers command-line tools to simplify, clip, and reproject large datasets efficiently. These can be run directly from the command line or integrated into a script for batch processing.
Effect: GDAL can handle large datasets more efficiently and can be used for preprocessing before loading the data into QGIS.
Summary
By simplifying your dataset, you reduce the computational load on QGIS and QSWAT, leading to fewer errors and smoother processing. Start by reducing the DEM resolution, clipping to your area of interest, and simplifying vector geometries. These steps will help make the dataset more manageable without compromising the accuracy needed for your analysis.
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I am trying to add 3 fragments to a vector. All 3 fragments are different sizes (125 bp, 4578 bp, 1445 bp). But I can't reach the correctly combined plasmid. I use 1 uL of each fragment and backbone, and I add the total (4uL) of gibson reaction (2x concentration). I tried chemical transformation and electroporation to send my gibson reaction to the cell and I can't observe any colonies. What am I doing wrong, what should I do?
Let me share a quote from my own essay:
"Dynamic flows on a seven-dimensional sphere that do not coincide with the globally minimal vector field, but remain locally minimal vector fields of matter velocities, we interpret as physical fields and particles. At the same time, if in the space of the evolving 3-sphere $\mathbb{R}^{4}$ the vector field forms singularities (compact inertial manifolds in which flows are closed), then they are associated with fermions, and if flows are closed only in the dual space $\mathbb{R}^{4}$ with an inverse metric, then the singularities are associated with bosons. For example, a photon is a limit cycle (circle) of a dual space, which in Minkowski space translationally moves along an isotropic straight line lying in an arbitrary plane $(z,t)$, and rotates in the planes $(x,t)$, $(y,t)$." (p. 12 MATHEMATICAL NOTES ON THE NATURE OF THINGS)
Near linear algebra textbooks.
I used PCR to add a few nucleotides to the 3' end of a gene on a pc3.1 plasmid and produced a linear vector with 18bp overlap at 5' and 3'. Then I used DpnI to cut off the template chain, transformed the linearized vector into BL21, plated it, picked a single clone and continued to culture it in Kan-resistant LB culture medium, and then handed it over to a sequencing company. The sequencing company said that my bacteria did not grow when further expanded and could not be sequenced. So I extracted the plasmid and sent it for sequencing again, and it still showed no signal. I simply asked the sequencing company to continue testing the Kan resistance gene, and it still showed no signal. If it is because the antibiotics are degraded, then when I did the transformation, the negative control that did not transform any genes did not have any colonies, which shows that the antibiotics worked. I am very confused. If the plasmid was not successfully transformed, why can it grow in the resistance culture dish?
Vector systems are essential tools in genetic engineering used to introduce foreign genes into host cells for purposes like gene cloning, gene expression, or therapeutic applications. The choice of vector system significantly influences both the efficiency and safety of these processes.
In quantum fluids the phase of a wavefunction is smooth and can be represented by a topological manifold of genus 0, the velocity creates a vector field over this manifold. Then can the hairy ball theorem be directly applied to state that there must exist a point where the vector field creates a vortex, showing a purely mathematical reason for the formation of vortices?
Hello, I am unable to file any plasmid map of the pHAL vector. I have searched addgene and other vector databases but still couldn't find anything, can anyone help me with this? Thanks.
Hi all,
Is there anyone who have the experience of solving nonlinear eigenvalue problem? I meet a special question.
The question is to solving the eigenfunction:
H(x) x=a x
where a is a real number, x is an eigenvector while H(x) is a matrix depended by x. The aim is to find a vector x and H(x) satisfying the equation which make the eigenvalue a minimum. In this question, H is far from a Hermitian in most case. And this question is not similar to conventional Kohn-Sham equation where most of the eigenvectors contribute to the construction of H. In contrast, in present question, H(x) may have many eigenvectors but, it depends on one of its eigenvector (x) only.
The method I try is self-consistent field method, i.e. determine a "temporary" H by a random or guess x, and by solving this temporary H a new x is obtained, and from new x a new H can be determined, etc. Currently, a major problem is that the matrix is not Hermitian, and therefore no real eigenvalue and corresponding eigenvector can be selected to define new "H". Since the target is to find a real number a and vector with real number elements, I hope that any temporary H can be composed by real number as well.
Does anyone have some idea about solving such a question? Any suggestion will be appreciated.
Thanks!
The question "What are the most common challenges in selecting an appropriate vector for cloning a large gene?" addresses the complexities involved in choosing the right vector for successfully cloning and expressing large genes in various host organisms.
Error 1:Invalid setting for output port dimensions of 'fccu/Mux6'. The dimensions are being set to 1. This is not valid because the total number of input and output elements are not the same
Error2:Error in port widths or dimensions. 'Input Port 1' of 'fccu/Zero-Order Hold' is a one dimensional vector with 1 elements.


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!
Dear all,
I have tried to use a mammalian Expression Vector System to overexpress a protein of interest in AGS cell line. Cells were selected based on puromycin resistance conferred by successful vector integration. However, despite successful antibiotic selection, I haven’t detected overexpression of the desired protein in this cell line. This has now happened for two different vectors encoding different target proteins.
Does anyone have any tips to reinforce the expression of the desired proteins or a possible explanation for this?
Thank you in advance,
Andreia Peixoto