Molecular Biology - Science topic

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Hello Md Fahmid Hossain Bhuiyan

No, you cannot perform TaqMan assay using average regular use PCR master mix.

If you wish to perform PCR using the regular PCR mix, the assay is no longer called TaqMan Assay because the defining feature of a TaqMan Assay is the probe. This small piece of DNA matched to the DNA template being measured has two special molecules attached, a fluorescent reporter dye (R) and a quencher (Q). While both molecules are attached to the probe, the fluorescence of the dye is suppressed by the quencher. These probes bind to the template DNA after it has been denatured into single strands but before it has begun duplicating, making sure that all duplications of the template interact with the probe.

During the PCR reaction, Taq DNA polymerase extends the primer through the polymerase activity, as it approaches the probe it displaces the probe and cleaves it through the 5′ to 3′ exonuclease activity. This separates the reporter dye and the quencher dye from the probe, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected in “real-time” directly by monitoring the increase in fluorescence of the reporter dye with an automated PCR system.

The assay which you would wish to perform is called two-step reverse transcription-polymerase chain reaction. In this assay, two enzymes are used namely, reverse transcriptase to produce single-stranded cDNA copies, which are then used as templates in an amplification reaction catalyzed by a thermostable DNA polymerase. This assay is the traditional method of RT-PCR in which the two synthetic reactions are performed separately and sequentially.

The TaqMan Assay is a real-Time PCR assay which detects the accumulation of amplicon during the reaction. The data is then measured at the exponential phase of the PCR reaction. The assay which you may plan to perform using average regular use PCR master mix is a type of conventional PCR using agarose gel which is not as precise as qPCR. By using the regular use PCR master mix, you cannot perform qPCR because for qPCR one requires the fluorescent reporter molecule such as fluorescent dye, a labeled oligonucleotide primer or probe such as (TaqMan Probe) for fluorescent detection which is monitored by the automated PCR system. Real-Time PCR makes quantitation of DNA and RNA easier and more precise than conventional PCR.

So, if you wish to use the average regular use PCR master mix, you need to perform the two-step reverse transcription-polymerase chain reaction and not qPCR.

Best.

doi: 10.7717/peerj.13489. eCollection 2022.. 2022 May 31;10:e13489.PeerJ

Prospective quantitative gene expression analysis of kallikrein-related peptidase KLK10 as a diagnostic biomarker for childhood acute lymphoblastic leukemia

Shwan Majid Ahmad 1, Basima Sadq Ahmed 2, Karzan Ghafur Khidhir 3, Heshu Sulaiman Rahman 4

Affiliations collapse

Affiliations

This article is indexed in pubmed.

For more entries, you have to contact the publisher directly; there is no automatic indexing, with respect to RG.

Certainly, the length and sequence of the 5' untranslated region (5'UTR) can potentially influence alternative splicing, as changes in the 5'UTR can alter the accessibility of splicing regulatory elements, affect translation initiation through the Kozak sequence, and impact the secondary structure of the mRNA. Additionally, the use of a strong promoter like pCMV can lead to high gene expression levels, potentially influencing spliceosome assembly and kinetics of RNA processing. In your case, the observed splicing pattern in the pcDNA3.1 vector might be a consequence of the long 5'UTR and strong promoter, which could be introducing or disrupting splicing regulatory elements. Further experiments involving different promoters and 5'UTR modifications are necessary to elucidate the specific mechanisms underlying the observed splicing differences.

It will be classic but you mentioned using a 30-minute reaction time, consider extending it slightly, as sometimes longer incubation times can improve RPA results, especially when using DNA extracted from complex samples. While 400 nM is a typical primer concentration for RPA, you can try titrating the primer concentration to optimize the reaction. Sometimes, higher or lower concentrations may work better depending on your specific setup. While the primers worked well in PCR, they might not be optimized for RPA. You could consider redesigning the primers specifically for RPA conditions. RPA primers may have different requirements, such as longer lengths (usually 30-35 bases) and specific GC content. First things that come to my mind. Good luck.

Dear Farah, I am struggling with the same issue.. Could you please tell what worked for the isolation of your Fosmid DNA?

Dear Igor, here is what I could find:

original depositor: J. B. Waterbury << (I) CCAP << (I) Ernst-Moritz Arndt-University

Original name designator: Myxosarcina chroococcoides; Chroococcidiopsis thermalis,

Source! Soil sample, near Greifswald, East Germany

Ref: Komárek, 1972; Waterbury & Stanier, 1978; Rippka et al., 1979

PS: I am to answer your email very soon...

Since there is no thesis writing universally in undergraduate level probably it must have meant long essay type project writing !

Hi Deekshitha .M

Do you wish to publish only in open-access journals? These days most journals offer a transformative publishing option, wherein you can choose to publish under a subscription model and you do not have to pay any money.

Elsevier, Springer, Taylor & Francis, Wiley- all these publishers have a hybrid mode of publication and you have to pay only if you choose to publish in an open-access model. These publishers also have a journal suggester/finder page where you just have to put your title, abstract, and keywords and you will be suggested relevant journals. There you can select the journal based on the scope of the journal and opt for the subscription model when submitting, so if in case the paper gets accepted then you don't have to pay at all.

Example: https://journalfinder.elsevier.com/

Best!

My Dear you apply for Canada visa and jobs along with resume.i can say that you can get there higher education with scholarship as you are scholar.

Very happy for your valuable open letter.i do not know in your united Arab Emirates citizenship.

Ok proceed.

RAM= 32 GB or higher

Processor= Intel core i7 or higher

High-end GPU instead of CPU

Linux OS

I would suggest using a workstation instead of a laptop.

Just to update, I've successfully transferred the small proteins / peptides from 20% acrylamide 1.5mm gel to a PVDF membrane using 250mA for 45 minutes. In case anyone googles this question :)

With the progression of the detected case of desminopathy, the appearance of viruses and gram-negative rods was established, there was an excessive bacterial growth of the fecal microbiota with a pronounced increase in transient microorganisms, an increase in endotoxin. The results are presented in the article: https://www.researchgate.net/publication/372952519_CHANGE_CHARACTERISTICS_IN_SALIVA_AND_FECES_MICROBIOTA_OF_A_DESMINOPATHY_T341P_PATIENT

Bonjour,

Pour le moment tous nos projets sont pris par les doctorants.

Cordialement. M. SIDQUI

Abhijeet Singh Thank you for your response and mentioning my earlier post! My belief is that researchers would know tools that are missing based on the fact that they would run into such problem often during their research. If there is some manual analysis task that researchers can automate, I believe that PeptiCloud can be the perfect platform to develop and make those tools publicly available. (For instance, PeptiCloud has a unique feature that allows users to further alter codon sequence of each amino acid after codon optimization with respect to a specific bacterial strain). With that being said, if you could check out PeptiCloud for yourself and see if anything could be added or improved, that would be greatly appreciated!

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.

Thanks Wolfgang, I appreciate the detailed answer!

Dear Dr. Saroj Kumar Khan

Yes, I agree with you that aggregation of protein is bad and is a cause of neurodegenerative diseases like Parkinson and Alzheimer's. Unfortunately, the mechanism explaining protein misfolding and protein aggregation, is not clearly understood at the molecular and cellular level.

It is well understood that functional proteins must pass through a quality control process in terms of folding to perform various physiological functions like catalysis, cellular transport, signal transmission and regulation, etc. However, there are a variety of structural and environmental factors that influence this process negatively. If you would be interested, I would like to list a few of them.

1. The protein structure, both the primary and secondary structures are most important for the physical and chemical characteristics. However, it could also be responsible for aggregation. For instance, the position and the number of hydrophobic amino acid residues in proteins may influence the aggregation behavior. The most common types of secondary structures, the α helix and the β pleated sheet may also have a role to play in protein aggregation.

2. Mutations play a determinative role in protein aggregation, and they may dramatically alter solubility, stability, and aggregation tendency of proteins. For instance, thermally stable proteins may change its stability even with a point mutation in its structure.

3. Post translational modification, especially phosphorylation plays a significant role in neurodegenerative diseases. For example, Alzheimer’s associated with tauopathy due to aggregation of the tau protein. In the brain, tau protein is found in neurons, and it can be phosphorylated with kinase enzyme. Thus, formation of aberrant tau aggregates accumulates in neurons, thereby exerting their toxic effects causing neuronal loss and synaptic alteration.

4. It is well-known that oxidative stress can cause protein oxidation, in particular, free radicals and ROS. Toxic free radicals can convert proteins into aggregation forms or proteins can be aggregated by conformational changes.

5. Environmental pH plays an important role in protein aggregation due to changes in net charge on protein. At low pH proteins may show aggregation tendency.

Best Wishes,

Malcolm Nobre

Your analogy is very interesting, dear colleague.

Although the main cause of any form of myofibrillar myopathy is a violation of the structure of the protein components of sarcomeres caused by genetic mutations, why not assume that due to mutations, the sensitivity of the postsynaptic membrane of myofibrils in myofibrillar myopathy to acetylcholine may also be impaired.

Hello Astelia Francois

Philip G Penketh is correct.

The Dalton symbol Da, is also sometimes used as a unit of molar mass, with the definition 1 Da = 1 g/mol.

Therefore,

1Da= 1g/mol

1 kDa = 1000 g/mol

So, 18kDa = 18000g/mol

Now

18000 g ---- 1M ---- 1L

18g----------1M -----1ml

18000mg --- 1M ---- 1ml

The protein concentration is 0.6mg/ml.

Therefore,

0.6mg x 1M / 18000mg = 0.0000333M = 0.0333mM= 33.3μM.

So the protein concentration (in μM) as per the size (18kDa) and concentration (0.6mg/ml) is 33.3μM.

Best.

I'd try isopropanol to remove the formamide. Should be more efficient than ethanol (at least from experience with precipitating nucleic acids from aqueous solutions). Basically, any solvent that is partially miscible with formamide should do the job, as long as the RNA doesn't dissolve in it. So, in the end, it will precipitate.

Yes, antibodies for immunofluorescence of frozen sections and cells can be shared. Antibodies used in immunofluorescence experiments might be shared among researchers or colleagues in order to conduct comparable experiments or validate results. Sharing antibodies enables replication and validation of findings, fostering scientific transparency and collaboration in the research community.

What I usually do is after activating PVDF membrane with methanol for 2-5 minutes, use Western-Blotting transfer buffer (usually is Tris/Glycine buffer containing 20% methanol) to wash the PVDF membrane for 5 minutes before do the transfer.

Thank you all for your answers.

Ammonium Sulphate precipitation works and may help resolve the particles by size. Note that the salt itself may affect the stability of the particles. In my hands, a 45% cut contained TMV CP monomer and multimers up to ~300k( analysed with boiled, reduced SDS-PAGE). I have read elsewhere that a 15% saturation is enough to precipitate rods.

Animal testing You can contact us This is our area of expertise

Philippe Paget-Bailly I believe that the switch from -80°C storage to -70°C storage for environmental reasons is a valid and sensible decision. The commonly used guideline of -80°C storage temperature originated from the technical limitations of cooling fluids used in the past, rather than being based on the long-term stability of samples.

The primary motivation for switching to -70°C storage is the significant reduction in energy consumption. It has been observed that the energy required to achieve and maintain temperatures lower than -70°C increases exponentially. By transitioning to -70°C, laboratories can potentially reduce energy costs by up to 30% compared to using -80°C freezers. This reduction in energy consumption aligns with the growing emphasis on sustainability and environmental responsibility.

The decision to switch temperature settings should be supported by scientific evidence and careful consideration of the specific samples being stored. The resources provided in the discussion include studies and information sheets that discuss the stability of various samples at different storage conditions. These resources can help researchers assess the impact of the temperature change on the integrity and longevity of their specific samples.

It is important to note that while the change in storage temperature may not directly affect the stability of the samples, researchers should ensure that the new storage temperature is within an acceptable range for maintaining sample quality and functionality. Factors such as the nature of the samples, intended duration of storage, and any specific temperature requirements should be taken into account.

In conclusion, the decision to switch from -80°C to -70°C storage for environmental reasons is a valid choice that can potentially reduce energy consumption without compromising the stability of samples. It is important for researchers to evaluate the specific requirements of their samples and consider the available scientific evidence when making such a transition.

After earning your Ph.D. in biology, you may seek a postdoctoral job in computational biology if you meet the prerequisites for the position. Computational biologists come from a wide range of disciplines, including biology, genetics, biochemistry, and other relevant topics.

Dear Soufiane Rabbaa

Add a non template control (NTC) in one PCR vial and test it.

A non template control is leaving the sample without cDNA. Usually NTC is used to check whether your cDNA is contaminated or to check primer-dimer formation. Here you can use the NTC to rule out which parameter (template quality, primer,reagents,dye) is to be rectified and troubleshooted.

All the best

Yes, there are a few studies that have investigated the effects of centrifugation on RALA peptide vector complexes. In general, these studies have found that centrifugation can lead to aggregation of the complexes, which can result in larger particle sizes. This is likely because the centrifugal force causes the complexes to collide with each other, which can damage the complexes and cause them to aggregate.

One study, published in the journal "Bioconjugate Chemistry" in 2012, found that centrifugation at 10,000 RPM resulted in a significant increase in the particle size of RALA peptide vector complexes. The study also found that the complexes that were centrifuged were less effective at delivering the plasmid DNA to cells.

Another study, published in the journal "Molecular Pharmaceutics" in 2013, found that centrifugation at 10,000 RPM resulted in a decrease in the transfection efficiency of RALA peptide vector complexes. The study also found that the complexes that were centrifuged were more likely to aggregate.

These studies suggest that centrifugation can have negative effects on the properties of RALA peptide vector complexes. Therefore, it is generally recommended to avoid centrifuging these complexes unless absolutely necessary.

If you do need to centrifuge RALA peptide vector complexes, it is important to use a low centrifugation speed (e.g., 5,000 RPM) and a short centrifugation time (e.g., 5 minutes). You should also avoid resuspending the pellet after centrifugation.

It is also important to note that the effects of centrifugation on RALA peptide vector complexes may vary depending on the specific protocol that is used. Therefore, it is important to experiment with different centrifugation conditions to determine the optimal conditions for your specific application.

Thanks for all your inputs

Hello Hemn Abdalla Omer

Do you mean you are looking for a suitable journal for your work in this topic?

In my opinion, kit tubes should be fine. If adherence is the problem, you can short-spin it after adequate tap mixing.

Hope, it helps.

Considering the necessity of growth factors for cells even for a few minutes, I usually use medium with fbs 2%, but serum-free medium is not recommended.

Don't forget that the transcription start site might be very far away from the ORF, especially if the ORF is part of an operon. So don't just look immediately upstream from the ORF.

There could be several possible reasons why no bands are observed in the PCR amplification of 16S rRNA of E.coli genes:

These video playlists might be helpful to you:

The SV40 (Simian virus 40) promoter is a strong viral promoter commonly used for driving gene expression in various experimental systems. While the presence of a TATA box upstream of the transcription start site is a common feature in many promoters, the SV40 promoter is unique in that it lacks a canonical TATA box.

The SV40 promoter utilizes an alternative mechanism for transcription initiation called the "TATA-less" promoter. Instead of relying on a TATA box, it utilizes other elements and transcription factors to initiate transcription. The absence of a TATA box in the SV40 promoter does not necessarily impair its ability to drive gene expression.

Therefore, in your current cloning strategy where the transcription start site is located just 8bp from the end of the SV40 promoter, it is likely that the expression can still occur without the presence of a TATA box. The SV40 promoter contains other regulatory elements and transcription factor binding sites that can facilitate transcription initiation.

However, it's worth noting that the exact transcriptional activity may depend on the specific context and the downstream sequence elements present in your plasmid. Experimental verification, such as measuring the expression levels of your gene of interest, can help confirm the functionality of the modified SV40 promoter in your specific system.

To perform a primary culture of non-parenchymal liver cells from mice, it is essential to choose the appropriate medium, determine the percentage of fetal bovine serum (FBS) to use, and decide on the necessary supplements. It is important to note that specific protocols may vary based on the intended application and the preferences of your laboratory.

For the medium, a commonly used choice for primary cell cultures is Dulbecco's Modified Eagle Medium (DMEM) or RPMI-1640. Both media are widely available and suitable for the growth of liver cells. The selection of the medium may depend on the specific requirements of your experiment or the protocols followed by your research group.

Regarding the percentage of FBS, a common range is between 5% to 10%. The choice of the exact percentage depends on the specific cell type and experimental conditions. It is advisable to optimize the FBS concentration based on the viability and growth characteristics of the non-parenchymal liver cells in your particular experiment.

As for supplements, commonly added components include L-glutamine, penicillin-streptomycin (antibiotics), and non-essential amino acids. The recommended concentration of L-glutamine is typically 2 mM, while the antibiotics are generally added at concentrations of 100 units/mL of penicillin and 100 μg/mL of streptomycin. Non-essential amino acids are often added at a final concentration of 1% or as specified by the supplier.

Additionally, considering the variability in experimental conditions, it is recommended to perform optimization experiments to ensure the optimal culture conditions for your non-parenchymal liver cells.

You've to be ble to analyse 3-D characteristics of both receptor & ligand and bond attractions which is very complex info, only known by very few highly trained chemists with high salaries and million usd bonuses work in pharma industries. But be sure almost nobody teaches these info to anyone since millions of usd gain, therefore you have to learn yourself if you are capable enough to learn !

I should still work, because the water should be cool even the ice was melt overnight.

Yes, your reading is correct. The article implies that there is potential for future nucleotide variations within the conserved restriction sites that are located in variable regions of the 16S rDNA gene.

The possible mechanisms for such variations are mutations, insertions, deletions, or recombinations, which can occur spontaneously or as a result of exposure to environmental factors, such as UV radiation, chemicals, or antibiotics. These changes can accumulate over time and result in differences in the sequence and/or length of the conserved restriction sites, leading to the generation of different band patterns upon restriction digestion.

The time frame for such variations can vary depending on the bacterial species, its population size, its growth rate, and the selective pressures it faces. Some bacterial species have high mutation rates and/or frequent horizontal gene transfer events, which can result in rapid evolution and diversification. Others have lower mutation rates and/or stable environments, which can lead to slower evolution and conservation of certain traits. However, even slow evolution can accumulate changes over time, and it is difficult to predict the exact time frame for future nucleotide variations within conserved restriction sites.

Regarding your edited question, the frequency of new 16S rRNA gene variants in bacterial species can also vary depending on the factors mentioned above. Some bacterial species have high genetic diversity and high rates of recombination and horizontal gene transfer, leading to frequent emergence of new variants. Others have low genetic diversity and low rates of recombination and horizontal gene transfer, resulting in slower emergence of new variants. However, the 16S rRNA gene is generally considered to be a stable and conserved marker for bacterial identification and classification, and many conserved regions within this gene are used as targets for PCR amplification and sequencing.

These video playlists might be helpful to you:

Hi Robert! Coming back in the past, which were your conditions?

Thanks!

Bertrand Cornu Can Kiessling Audrys G. Pauža Mohamed Khashan Dino Santos Matias Thank u all. After many trials I have decided to use single temperature. I have to admit that still qPCR experiment is not so objective to me (changes according to conditions very easily). However, since everyone use the same method, and what matters is to compare the mrna level for the same protein, I guess it is fine.

The Vienna Atomic Line Database (VALD) is a comprehensive database of atomic and molecular transition lines. It contains over 50 million lines for over 100,000 atoms and molecules.

The National Institute of Standards and Technology ASD (The NIST ASD) is a database of atomic and molecular spectral data. It contains over 10 million lines for over 80,000 atoms and molecules.

If the database contains lines that match the molecule's signature, then you will be able to identify the molecule in the spectrum.

Figure 1 shows Imatinib disrupts the protein-protein interactions of Bcr-Abl with some, but not all interaction partners.

For cDNA synthesis, high-quality RNA with an A260/A280 ratio of 1.8 to 2.2 is ideal, and a minimum concentration of 1 μg/µL is usually recommended. For Real-Time PCR, the optimal RNA concentration is highly dependent on the gene of interest, and can range from 1 ng/µL to 500 ng/µL.

The efficiency of Gibson cloning is higher than that of Golden Gate. Gibson cloning is a one-step assembly method that uses a DNA ligase enzyme to join two or more DNA fragments together. It is highly efficient, with reported success rates of up to 95%. Golden Gate cloning is a two-step assembly method that relies on a restriction enzyme and a DNA ligase to join two or more DNA fragments together. This method is less efficient, with reported success rates of up to 80%.

I recently sequenced pHIT60 via https://www.plasmidsaurus.com. In case anyone is still looking, Iv'e attached the gbk file!

No, introns are not considered in designing primers for full-length gene regions in the DNA template. Primers are designed to amplify only the exonic regions of the gene.

Hello Hui Yee you could also try to use PICO to detect your MyoD. They claim to be much more sensitive compared to Co-IP/WesternBlot workflows. Here is an explanation of the technology:

Arthur Kornberg, For the Love of Enzymes: The Odyssey of a Biochemist, is mainly focused on the discovery of enzymes involved in nucleic acid acid replication.

Indumati Sharma Did you figure out the working temperature for this? I am going to perform this and am quite confused which temperature should I prefer? And can you please also specify the blank you used?

Agree that just because it's DNA-binding, that doesn't mean it can't be secreted too. But if you are sure that it is not secreted, necrosis-type cell is the only way I can think of for it to get into serum. Also, are you sure the protein is only expressed in monocytes?

As for your result, are you confident your protocol for making the cell lysate does enough to preserve protein stability? Cell lysates can be fickle in that regard.

Another potential issue are posttranslational modifications. If the secreted form has different PTMs than the intracellular one (which is common), this might affect antibody affinity.

Andrew Paul McKenzie Pegman Hi! thanks for replying. I earlier read about mucilage and as I understand mucilage is the collective term used for the secretion by seed during germination. I am looking for specific term for these cells in image. These are single free floating round cells and these are viable even when they are away from the seed coat.

Dear Sir you may try the protein separation with foam fractionation coloumn. it would suppose to help you

I highly recommend you to focus on your education and understanding the basics and fundamentals and not to spam here by posting questions and answering yourself.

Further, since you don't have the proper education to understand what software can be used for what it is totally illogical to talk about bioinformatics tools.

The answer to your question should be long. The disease resistance is usually conferred by a major gene while the crop yield is conferred by multiple minor QTLs/genes. Also, there are different approaches to isolating a gene of interest. There are several main steps to isolate the disease-resistance gene using a map-based approach.

1. Perform QTLs analysis using a small population like RILs, NILs, or DH that were derived from two parents carrying opposite genotypes.

2. Screen the plant carrying recombinants within the QLT of interest.

3. Develop internal markers to fragment the genetic window

4. Narrow down the genetic window based on the combination of genotype and phenotype of plants carrying recombinants.

5. Identify the list of candidate genes within the final genetic window

6. Validate the candidate genes to identify the actual gene conferring the trait of interest (using mutant analysis or gene transformation).

Good luck!

I agree with both Gregory and Era. Try washing your sponges and transfer equipment thoroughly. I would also be careful with your membrane regarding containers etc you use for washing / staining.

Hi all,

Thank you for your answers,

I did a restriction digest with enzymes that cut multiple times and indeed, this plasmid has recombined in all sorts of ways except the one I was planning on...

I don't know if any of you have practiced recombineering before, but if you have I would really appreciate your advice regarding how to reduce unwanted recombination events in this type of cloning.

I am using an L-arabinose inducible plasmid for the λRed system. Are NEB10betas good cells for these protocols or maybe Stabl3 would be a better option? Also, would co-electroporating my plasmid at very low concentrations and the linear dsDNA into E. coli (which contains the induced λRed system-plasmid) help in avoiding these undesired recombinations?

Any other thoughts or help on how to avoid this?

Thanks!

Did you find the solution? I am in the same trouble.

Daniil ( Даниил ) Александрович Fedorov ( Федоров )

Difference equations can be useful for modeling discrete events in biological systems, such as the discrete stages of a cell cycle or the discrete events of a neural network firing. However, they may not be ideal for modeling continuous processes, such as the dynamics of a chemical reaction or the movement of a fluid through a network of vessels. In these cases, continuous models, such as differential equations or agent-based models, may provide a more accurate representation of the underlying biology.

One limitation of difference equations is that they assume that the changes in a system occur in discrete steps, and do not account for small changes that occur continuously over time. Additionally, they may not be able to capture the effects of small perturbations in the system, which can have a significant impact on the overall behaviour of the system.

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.

Essien Archibong Okon I worked in commercial plant disease diagnostic lab under the same roof with Plant tissue lab from 2012 till 2021. Finally, we found such location incompatible with stable production of pathogen-free meristemic plants. Do not repeat the same mistake.

Ho Okay, I've effectively seen it in the genome of DH5alpha but I did not find the genome of TOP10. Thanks a lot. In fact the insertion take place in the replication regulation area. May be it can influence the copy number to limit the toxicity of the cloned protein (a fluorescent protein). I will check on the website of Scarab Genomics to order a first batch of these clean E.coli ! Thanks again Alexandra !

What origin of replication are you using? Rolling circle replication plasmids (especially with only a single stranded ori) like pWV01 tend to be much more unstable to secondary structure and short repeats than theta replicating origins such as pAMB1.

If you are talking about using the antibodies as the affinity matrix (such as in immunoaffinity chromatography), I would suggest that the way to avoid damage to the antibodies from proteases in a crude extract is to save the immunoaffinity chromatography for the last step in the purification, at which point the protease activity from the crude extract should be greatly reduced. Begin the purification with ion exchange, hydrophobic interaction, and/or gel filtration chromatographies. Use a protease inhibitor cocktail when making the crude extract.

Dear Geir Bjorklund, Duc M. Hoang, John Hildyard, thank you very much for your answers and recommendations!

To prevent recombination events in your Lactococcus lactis strains, you can consider knocking out genes involved in homologous recombination. The genes involved in the homologous recombination process in Lactococcus lactis include recA, recB, recC, and recD. By disrupting these genes, it is possible to prevent homologous recombination and reduce the likelihood of recombination events in your plasmids.

Additionally, you can also consider reducing the growth rate of the bacteria during your culture phase, which may help to reduce the rate of recombination events. This can be achieved by adjusting the temperature, pH, or nutrient conditions, or by modifying the composition of the growth medium to reduce the rate of bacterial growth.

Hope it helps