Molecular Biology - Science topic

Yes I am interesting, Researcher from Sudan work on oncology centre

Hello everyone! I am working on cloning plant genomic dna and I made primers usin snap gene for cds region. The amplified fragments are not the one I require. For instance, the cds region sequence is 1719 basepairs and the band on gel electrophoresis is around 3000 base pairs. I have used Phanta 2X Max polymerase. What could be the reasons can anybody please guide. The genomic dna is from legume.

Hi there,

A 3'H instead of the 3'OH is 100% efficient to block extension! This is the principle of Sanger sequencing...

The oldest relevant reference in the OED is to a paper by Gamow and Yčas from 1955, namely Statistical Correlation of Protein and Ribonucleic Acid Composition.

Shler Ali Khorshed Thank you very much for the detailed information!

Coating a microplate with viruses for a whole-virus enzyme-linked immunosorbent assay (ELISA) requires precision and adherence to a standardized protocol to ensure reproducibility and accuracy. Here is a detailed, step-by-step guide on how to coat a microplate with viruses for this purpose:

By following these detailed steps, you ensure that the virus is properly adhered to the microplate, maximizing the sensitivity and specificity of your whole-virus ELISA. Each step, from the preparation of the virus to the final storage of coated plates, is designed to maintain the functional integrity of the viral antigens and provide reliable, reproducible assay results.

Check out this protocol list; it might provide additional insights for resolving the issue.

It is hypothetical since its existence is presumed based on the transcriptomic data rather than being directly detected by proteomics or WB. You can try based on some apriori knowledge about it as a lead molecule or entirely randomly. Nothing wrong with that. Also, 'polypeptide' suits better for an amino acid chain this short.

The forecast suggests that molecular-based methods will increasingly become standard procedures in monitoring antibiotic resistance. These methods offer advantages such as speed, accuracy, and the ability to detect multiple resistance genes simultaneously. As technology advances and becomes more accessible, these methods are likely to become more widely adopted in clinical and public health settings for monitoring antibiotic resistance trends and guiding treatment decisions.

Using primers longer than 100 base pairs (bp) for cloning purposes is not a common practice, but it can be necessary for certain applications, such as incorporating large tags, mutagenesis of multiple sites simultaneously, or cloning sequences with high secondary structure. Long primers allow for the introduction of complex modifications and can facilitate the assembly of sequences with precise control over the genetic architecture. However, working with long primers presents unique challenges and considerations.

Design Considerations

Synthesis and Purification

PCR Optimization

Cloning Strategy

Troubleshooting

Conclusion

While using primers longer than 100 bp for cloning is challenging, it is feasible with careful design, high-quality synthesis, and optimization of PCR conditions. These primers offer flexibility for complex cloning projects but require meticulous planning and execution to ensure success. Always verify the final construct sequence to confirm that the intended modifications have been accurately incorporated.

Take a look at this protocol list; it could assist in understanding and solving the problem.

NaI can be replaced by KI, since iodide reduce Tm of protein and cause instability of folded protein, which had its purpose for protein denaturation, but potassium may increase the pH. For sodium N-lauroylsarcosine, as long as sodium lauroyl sulfate provide a membrane disruption ability, then the DNA should be released as the same way as original protocol. All your reagents should be in tris-HCl and adjusting to a final pH 8, because in this pH tris has its best buffer ability.

Best

A cell line that does not express both iNOS (inducible nitric oxide synthase) and CD163 (cluster of differentiation 163) could be a cell line derived from a tissue or cell type that does not typically express these markers under normal conditions.For example, many cancer cell lines, such as HeLa cells or MCF-7 cells, may not express iNOS or CD163 unless they are specifically induced to do so under experimental conditions. Additionally, certain immortalized cell lines derived from non-immune tissues, such as fibroblasts or epithelial cells, may not express these markers.However, it's important to note that the expression of iNOS and CD163 can vary depending on the experimental conditions and the specific context in which the cells are studied. Therefore, it's always a good idea to confirm the expression profile of a cell line using experimental techniques such as immunostaining, Western blotting, or flow cytometry.

The difference between using an IRES (Internal Ribosome Entry Site) and an IRES2 lies in their efficiency and specificity in driving gene expression in a bicistronic mRNA.IRES (Internal Ribosome Entry Site): IRES is a sequence element within the mRNA that allows ribosomes to initiate translation internally, bypassing the requirement for a 5' cap structure. When an IRES is present in a bicistronic mRNA, it enables translation initiation of the downstream gene even if the ribosome is still translating the upstream gene. However, IRES elements are generally less efficient than cap-dependent translation initiation, leading to lower expression levels of the downstream gene compared to the upstream gene.IRES2: IRES2 is an improved version of IRES that has been engineered to enhance its efficiency and specificity. IRES2 sequences have been optimized to increase translation initiation rates and reduce leaky scanning (initiation at inappropriate start codons). As a result, IRES2 elements typically lead to higher expression levels of the downstream gene compared to traditional IRES elements.In summary, while both IRES and IRES2 facilitate translation initiation of downstream genes in bicistronic mRNAs, IRES2 generally offers higher expression levels due to its improved efficiency and specificity.

@Robert Adolf Brinzer The question is, what is the possible structural and functional role of newly discovered SIRC (Short Interrupted Repeat Cassettes) elements in plant genome. The point is that in bacterial genomes there are CRISPR cassettes which are looking similar to SIRC but have nothing common.

Dear Esteemed Colleague,

Greetings. I trust this message finds you deeply engaged in your research and seeking answers to complex questions within the realm of genetics and molecular pathology. Your inquiry regarding the potential role of infection in causing desmin mutations in myofibrillar myopathy is both intriguing and indicative of a keen scientific mind exploring the multifaceted nature of genetic disorders.

To address your question with the precision and clarity it deserves, it is crucial to first understand the nature of myofibrillar myopathies and the role of desmin within this context. Myofibrillar myopathies are a group of neuromuscular disorders characterized by the progressive weakening of muscles and the disintegration of muscle fibers at a cellular level. Desmin, a type of intermediate filament protein, plays a pivotal role in maintaining the structural integrity and function of muscle cells. Mutations in the DES gene, which encodes the desmin protein, are directly linked to certain forms of myofibrillar myopathy.

The genesis of these mutations, particularly those affecting the desmin protein, is primarily genetic, resulting from inherited or de novo mutations in the DES gene. These mutations lead to the production of an abnormal desmin protein, which disrupts the normal architecture of muscle cells, leading to the symptoms associated with myofibrillar myopathy.

Addressing the specific question of whether an infection could cause desmin mutations, it is essential to differentiate between the origins of genetic mutations and factors that may exacerbate the phenotype of a genetic disorder. Genetic mutations, including those affecting the desmin gene, arise from alterations in the DNA sequence. These alterations can be inherited from parents, occur spontaneously during DNA replication, or be induced by certain environmental factors, such as exposure to specific chemicals or radiation. Infections, while capable of causing a wide array of health issues, do not directly induce genetic mutations in the DNA sequence of the genes like DES. However, it is conceivable that certain infections could exacerbate the clinical manifestations of myofibrillar myopathy in individuals already predisposed or carrying a desmin mutation, by stressing the muscular system or triggering inflammatory responses that may further compromise muscle function.

In conclusion, while infections can have significant impacts on overall health and may interact in complex ways with genetic disorders, the mutations in the DES gene that cause myofibrillar myopathy are not directly caused by infections. The mutations are genetic in origin, and the relationship between infections and the severity or progression of myofibrillar myopathy would be more accurately viewed through the lens of infection exacerbating pre-existing conditions rather than causing the genetic mutation itself.

I hope this elucidation addresses your inquiry comprehensively. Should you have further questions or require additional clarification, please feel free to reach out.

Warm regards.

This protocol list might provide further insights to address this issue.

Yes and no.

60°C is used for almost all reactions, but not all. For example, in my case, I amplified some genes for Gibson cloning, so my oligos have a high size (50/60bp). In these cases, the annealing step is not necessary and you just proceed to the extension step. The enzyme buffer permits annealing at 72°C. In all my reactions, I always had high amplification and I always followed the protocol parameters.

You just need to be careful with the correct design of your primers and with your DNA sample. Your sample needs to be as pure as possible, and you should use the amount recommended by the protocol in each reaction, which is 10ng if the plasmid gene is being amplified. When I used a bit more sample, the amplification didn't occur.

I do find it reliable, much more so than other enzymes I've used. We haven't had any problems with genes amplified with it and used in cloning.

Ruslan Kalendar

I added 3 M NaAc pH5 and the purification still didn't work. Still 0% recovery.

I used pH strips too. The pH is low but still 0% recovery.

Dear Colleague,

I trust you are doing well. In response to your request for suggestions on reasonably priced oligonucleotide synthesis services, both within India and internationally, I am pleased to provide a comprehensive overview aimed at facilitating your decision-making process.

When selecting an oligonucleotide synthesis service, consider the following criteria to ensure you receive the best value and quality for your research needs:

Before finalizing your decision, it may be beneficial to request quotes from multiple providers and evaluate any bulk order discounts or promotional offers that could further optimize your investment. Additionally, reaching out to your professional network for firsthand reviews and experiences can provide valuable insights into the reliability and quality of the services you are considering.

Should you have any further inquiries or require assistance in contacting these services, please feel free to reach out.

Best regards,

With this protocol list, we might find more ways to solve this problem.

AOA

Dear Mashal

I am interested in collaboration with you. Thank you.

Yes, you can adapt the protocol for preparing electrocompetent E. coli cells from DH10B to JM109. However, it's important to note that different strains of E. coli may have slightly different requirements for optimal transformation efficiency, so you may need to optimize the protocol for JM109 cells.

Here's a general outline of how you can adapt the protocol for preparing electrocompetent JM109 cells:

By following this adapted protocol, you should be able to prepare electrocompetent JM109 cells for your Gibson Assembly experiments. It's always a good idea to perform optimization experiments to determine the optimal conditions for your specific application.

Dear Colleague,

Among the structures listed — plasmid, pilus, capsule, and plasma membrane — the entity that contains genes for enzymes and antibiotic resistance is the plasmid. Plasmids are small, circular, double-stranded DNA molecules that are distinct from a bacterial cell's chromosomal DNA. They are capable of autonomous replication and often carry genes that may confer advantageous traits to bacteria, such as antibiotic resistance and the production of enzymes that degrade toxic compounds.

In conclusion, plasmids are the structures among those listed that contain genes for enzymes and antibiotic resistance, underscoring their significance in bacterial adaptation and survival, especially in environments with selective pressures such as antibiotics.

Yours sincerely,

Perhaps this protocol list can give us more information to help solve the problem.

Can you clarify the roles of the variables you mentioned? If one of them is a dependent variable, for example, which one is it? Thanks for clarifying.

Please clarify too what "a trait with continuous variables" means. Perhaps if you just said what the trait is (and what the continuous variables are), it would help readers to understand better. Thanks.

Dear Esteemed Colleague,

Following the completion of a two-way ANOVA, which serves to ascertain the effects of two independent variables on a dependent variable, as well as any interaction between these independent variables, your subsequent steps should be methodically oriented towards a comprehensive interpretation and validation of the results obtained. Here is a structured approach to guide your post-ANOVA analysis:

By following these steps, you will ensure not only the rigorous analysis of your two-way ANOVA results but also the meaningful interpretation and reporting of these results within the broader context of your research field.

Should you require further assistance or clarification on any of these steps, please do not hesitate to reach out.

Warm regards.

This protocol list might provide further insights to address this issue.

Desminopathy, also known as desmin-related myopathy (DRM), is a rare genetic muscle disorder that affects the protein desmin. Desmin is an essential component of the intermediate filaments that provide structural support within muscle cells. Mutations in the DES gene, which codes for desmin, lead to disruptions in the normal structure and function of muscle fibers.

The muscles you mentioned - Semitendinosus, Gracilis, and Sartorius - are often affected at the onset of desminopathy due to their specific characteristics and biomechanical roles.

1. Semitendinosus: The semitendinosus is one of the three hamstring muscles located in the back of the thigh. It plays a key role in knee flexion and hip extension. The semitendinosus muscle is frequently involved in desminopathy due to its high proportion of slow-twitch muscle fibers, which are more vulnerable to desmin-related abnormalities.

2. Gracilis: The gracilis muscle is a long, thin muscle located in the inner thigh region. It is involved in hip adduction and knee flexion. Similar to the semitendinosus, the gracilis muscle also consists of a high proportion of slow-twitch muscle fibers, making it susceptible to desmin-related abnormalities.

3. Sartorius: The sartorius muscle is a long, strap-like muscle that runs diagonally across the front of the thigh. It plays a role in hip and knee flexion and also assists in thigh rotation. The sartorius muscle is affected in desminopathy due to its similar composition of slow-twitch muscle fibers.

The predilection for these specific muscles in desminopathy may be attributed to their fiber type composition and the mechanical stress they experience during certain movements. However, it is important to note that desminopathy can affect other muscles as well, and the degree and pattern of muscle involvement may vary among individuals with the same genetic mutation.

It is advised to consult with a medical professional or genetics specialist for a more accurate assessment of muscle involvement and management of desminopathy.

Hi!

Please send

Email Address: [email protected]

A distance of 20 bp in DNA (I assume) has a length of 68 Angstrom. This should be close enough for a significant extent of FRET quenching by BHQ-1.

The behavior of FAM in this system could be surprising. It is likely to be at least partially quenched already due to interactions with the DNA, regardless of the BHQ. If the oligo is denatured, its fluorescence intensity could increase. In other words, for certain uses, the BHQ may be unnecessary.

Hello Chandra Kishore,

You may use density gradient centrifugation. In density gradient centrifugation depending on the density of the particles in the sample, similar substances will group together when exposed to rotational force. Because dead cells and cellular debris are fractured, they become less dense than living, healthy cells. Adding in certain separation reagents such as Ficoll can purify the sample by acting as a barrier that only one population can pass through.

For instance, in a 50 mL centrifuge tube, you may layer 18ml of your cell suspension onto 12ml of a Ficoll-paque combination. Centrifuge your tube for 15 minutes at 400x g. When centrifugation is complete, you will note that the live cells will collect at the interface and the dead ones will form a pellet at the bottom of the tube.

Another simple method as mentioned by Samir would include centrifuging the cell suspension at 150-200g for 10 mins. You may discard the supernatant which consists of cell debris and resuspend the cell pellet in fresh medium.

Best.

When performing a ligation reaction with one vector and two inserts, there are a few key considerations to increase the likelihood of successful ligation. Here are some recommendations:

1. Vector and Inserts Preparation: Ensure that the vector and inserts have been properly prepared and purified. This includes digesting the vector and inserts with the appropriate restriction enzymes, dephosphorylating the vector (if necessary) to prevent self-ligation, and purifying the DNA fragments from the digestion reaction using a DNA purification kit.

2. Insert Ratio Optimization: Determine the optimal molar ratio of vector to inserts. This ratio can vary depending on the specific experiment, but a standard recommendation is to use a two-fold molar excess of the vector compared to each insert. Adjust the amount of DNA accordingly to achieve the desired ratio.

3. Vector Control: Include a vector-only control in the ligation reaction. This control helps to identify any background self-ligation of the vector or potential contamination of the components.

4. Extended Incubation: Allow for a longer incubation time during ligation. Incubating the ligation reaction overnight at a lower temperature (such as 4°C) can improve the chances of successful ligation by giving the enzymes more time to bind DNA ends and create ligated products.

5. Optimal DNA Ligase Concentration: Determine and optimize the concentration of DNA ligase enzyme in the ligation reaction. It is recommended to follow the manufacturer's instructions or perform preliminary experiments to find the appropriate concentration that promotes efficient ligation without causing excess background ligation.

6. Ligation Enhancers: Consider using ligation enhancers, such as polyethylene glycol (PEG) or dimethyl sulfoxide (DMSO), to increase ligation efficiency. These additives can enhance DNA hybridization and enzyme activity, thus improving the likelihood of successful ligation.

7. Transformation and Selection: Transform the ligation reaction into competent host cells using an appropriate method (e.g., heat shock for bacterial cells). Following transformation, select transformed cells using selective media containing the appropriate antibiotic resistance marker present in the vector.

8. Verify and Validate: Perform colony screening, diagnostic PCR, or DNA sequencing to verify that the desired ligation product has been successfully obtained. This step is crucial to ensure that the correct inserts have been ligated into the vector.

By following these recommendations and optimizing the key parameters, you can increase the chances of obtaining successful ligation with one vector and two inserts. It is always advisable to consult relevant scientific literature or seek guidance from experienced researchers in your specific area of study to further refine your ligation strategy.

Try increasing the extension time during the PCR so it has time to go to the end. You can do an initial cycle at the lower temperature and then do the remaining cycles at the higher temperature for more stringency. Do you have a stop codon after your 6xHis tag?

Snapgene is fine to your need.

I really can't understand how you can deal with molecular biology with a light microscope !?

Tamoxifen free base seems most frequently to be given in corn oil i.p. in, for example, CreER conditional KO mice. We have routinely used tamoxifen citrate which can be formulated in aqueous vehicles for i.p. injections (such as 0.5% methycellulose). In our experience this protocol is much more humane for the mice, and the citrate salt can be dosed at between 5 and 20mg/kg, rather than the 100mg/kg or more frequently used for the free base. Oral dosing leads to lower bioavailability, and mice don't like tamoxifen food pellets and in some cases will refuse to eat them at all.

Centrifugation conditions for concentrating lentivirus can vary depending on the specific protocol and the equipment being used. However, I can provide a general guideline that is commonly followed:

Remember, these are general guidelines and can vary. It's crucial to follow the specific protocol recommended for your lentivirus preparation and the equipment you are using. Always ensure that you are working in a biosafety cabinet and following appropriate biosafety protocols when handling lentiviruses, as they are potentially biohazardous materials.

l This list of protocols might help us better address the issue.

Microbiome analysis and next-generation sequencing are recent topics. However, your choice of research topics should align with both the available lab facilities and your personal interests. All the best for your research work!

Yes, that’s correct! In strand displacement synthesis, the primer strand is extended using the template strand, and each nucleotide incorporated displaces the third strand that was annealed initially to the template.

The third strand is a short, single-stranded DNA annealed to the template strand and acts as a stabilizing agent for the displaced strand . The displaced strand is the second strand of the double-stranded DNA being synthesized.

More at:

- Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB | Nucleic Acids Research | Oxford Academic (oup.com)

-D-loop - Wikipedia

Check the electrodes. If you see them covered with whitish stuff, remove it with wet tissue or brush until the metal surface is exposed.

To main the Rosetta 2 (DE3) strain, do we have to grow them in any antibiotic? Chloramphenicol?

If you or others have not tested this antibody before then it's possible this is just a "bad" antibody that binds to multiple proteins. It's very common for antibodies to show different results than what is advertised by the company selling them.

I can't find much information about your protein marker, but you should make sure this marker is compatible with chemiluminescence. Ponceau staining will stain all proteins, so that's why you could see the marker protein there. But if the protein marker is not modified or your secondary antibody does not bind to the marker proteins, then you will get no chemiluminescence signal from the marker protein. You can read more about this at the bottom of this page: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/chemiluminescent-western-blotting.html

Hi Prem

There are several salt-free media for mammalian cell culture, including DMEM w/o NaCl, IMDM w/o Na/Cl, etc. However, salt-free media refer to the one without NaCl, they may still contain other salts such as KCl. These salts are often necessary for cell growth and function. Thus, I'm afraid there may be no completely salt-free media.

Your gel should have the exact same layout as your Western blot, because your Western blot is a transfer of your gel directly onto the membrane. You might not be noticing the right band on your gel. Maybe the band is very thin by eye with coomassie, or even barely visible, but it jumps out as a very major band with the antibody detection. This happens frequently. Most proteins studied are not the major protein expressed in the cell.

Do you have a different molecular weight ladder for Western and Coomassie stain? If you run the same ladder on both, that may clarify the situation.

Proteins do not necessarily run on the SDS PAGE gel according to their expected molecular weight from the protein sequence. There are several possible reasons for this, such as glycosylation and shape.

Hi Harshita

lot of possibilities, but the main ones are to go to the NCBI or UCSC database (for instance, just type NCBI XXXX YYYY UTR region, where XXXX is your bacteria and YYYY your gene) in google.

or just give the species and target in research gate...maybe someone could answer ;)

all the best

fred

When working with membrane proteins and performing Western blot analysis, it's essential to choose an appropriate loading control that is not affected by changes in membrane protein expression. Commonly used cytosolic loading controls, like beta-actin or GAPDH, may not be suitable in this case, as their expression levels can vary in response to changes in membrane protein expression.

Here are some options for loading controls when studying membrane proteins:

As for the availability of antibodies, it depends on the specific protein you choose as the loading control. Antibodies against some common membrane proteins, such as Na^+/K^+ ATPase or V-type H^+-ATPase, are commercially available from reputable antibody suppliers. Ensure that the selected antibody recognizes the appropriate epitope and has been validated for Western blotting.

Before finalizing your loading control strategy, it's crucial to conduct preliminary experiments to validate the stability of expression of the chosen loading control(s) under your experimental conditions. Additionally, consider consulting the literature or seeking advice from researchers with expertise in the specific membrane protein and experimental system you are working with.

I agree with the suggestions above. Also, is anyone else having the same issue and if so, what are any commonalities? Same SYBR? Same primers? Same QPCR machine?

My best guess would be your cDNA is not good. But, I did have a QPCR machine itself stop working one time (and I was the only user at the time). Double check your program too (sample setup and cycle parameters).

In certain DNA replication errors, particularly during template-switching events like strand slippage or misalignment, the resulting duplication can indeed be either in the same orientation (tandem duplication) or inverted (inverted duplication) relative to the original sequence. This can occur under specific circumstances and mechanisms during DNA replication.

In template-switching, which is often associated with replication errors like slipped-strand mispairing, the DNA polymerase temporarily dissociates from the template strand and switches to a nearby template strand before continuing replication. The orientation of the newly synthesised DNA strand and the template strand to which it switches can determine whether the resulting duplication is in the same or inverted orientation. Here's a basic explanation:

The specific circumstances and outcomes of template-switching events can vary, and they are influenced by factors such as the length and sequence of the repetitive elements in the DNA, the timing of the switch, and the precise mechanisms involved in DNA replication. These events can lead to genetic rearrangements and contribute to the diversity of genomic structures, which can have implications in terms of genetic variation and evolution.

Ryan Gourlie

Nusrat Malik Nusrat, for people beginning to learn the concepts all the intext citation interrupts the flow of the words and greatly lessens comprehension. its akin to looking at a cell phone while reading. You have to decide if want to teach the formalities of academic publications and if they're more important than comprehending a new idea.

@ Good morning

Thank you very much. I am going to test them.

Regards

Lyudmil Antonov Aaron Laud Yes, sirs, I ordered several specific primers and now I have got a standard strain of E coli O157... I hope I'll find the gap in the next trial of a PCR reaction

Hussain Ali Rzoqy surely sir, I was cultured and identified the isolates.. but I couldn't characterize between E coli O157 H7 and other species until now.

Many thanks for your helpful discussion and opinions.

Short answer - I would say yes. If an enzyme can't turn over products on any time scale, its turnover number would be 0.

I agree that if kcat = 0, then effectively your K_M is just the ratio of k_on and k_off, that is, it is equal to the K_D. If one was simply measuring binding of the substrate to the enzyme, I can definitely see that replacement of a residue critical to the mechanism, but not the binding and orientation of the substrate, would generate a variant that does not turn over product but still has a similar or identical K_D for the substrate. This says nothing about the turnover number (or lack thereof).

How would I show a reviewer that a binding-competent enzyme is catalytically inactive? I think what you have described is going above and beyond. Extending a reaction for 60 min at 100 x K_M to prove no activity exists feels a bit extreme. Even if the measured kcat is very tiny but not zero, it would be a moot point as this has absolutely no biological relevance.

If your wild-type enzyme has a kcat > 1 s^-1 like you mention, I think it would be reasonable to measure the initial rates of i) the wild-type enzyme, ii) the catalytic mutant, and iii) a matched enzyme-free reaction using the same reaction conditions for all on whatever time scale is appropriate for your enzyme and its relative rate.

With enough replicates, you can use appropriate statistics to compare the grouped samples. Showing that there is no statistical difference in the rate of reaction between an experiment with no enzyme at all versus a catalytic mutant would be a strong argument that no activity exists, i.e. kcat must equal 0.

You will see in this paper we use an LC-MS assay to detect the presence/absence of product in active site replacements as justification for a dead enzyme, in addition to a colorimetric assay where we did the above to justify activity as "n.d." We did not measure affinity to the substrate but the binding site is > 15 angstroms away in this case.

Hope this helps!

ACA

Hello Maria,

I normally do three steps cleaning to work with RNA related work. First i start with 70% ethanol, then RNAse zap and finally nanopure water (work area and pipettes). NP water is use specially on metal part of any equipment to avoid corrosion . I have seen people only using lot of 70%ethanol instead of RNAse zap.

Hope that helps :)

Hello,

The laws and means of financing differ from one country to another, the best thing to do is to first contact a laboratory in your country and ask them how they obtained the financing, after which you can see with the health authorities of your country to request possible assistance for the creation of your laboratory.

I work with human cell lines and often find that genes I'm studying are expressed as one of the predicted "XM" transcripts, so I would definitely include them in your primer design. However human genes have lots of isoforms, so for your specific organism maybe XM transcripts are less likely to be expressed.

For your second question, I would choose to design a good primer that doesn't cross an exon-exon junction. DNase treatment is usually pretty robust, but more importantly you should be including some "no RT" controls in your qPCRs, which will tell you if you get amplification from DNA.

If someone knows German and certifies with DAF exam, he/she may have incredible positive opportunities. Other than Germany there seems to be unfortunately no hope !

I am a rheumatologist. Would be more than interested to be a part of any project involving Musculoskeletal disorders

Upper gel:

In samples 3 & 4 there is obviously no or very little DNA.

In my opinion all other DNAs are not properly dissolved and the gel is overloaded; and you might have RNA contamination.

Ensure your DNAs are completely dissolved and as suggested by others determine the concentration and reload equal amounts.

And, as also suggested by others, you should always seek advice from your supervisor or a senior lab member.

Primer concentration is important in this experiment (usually in all amplification experiments). In addition to it,my question is whether the recombinases enzyme and sample in your experiment are working properly?

All the best

Sara Melo-Dias

Hi there,

These are strong constitutive promoters and terminators. Any combination should be OK... I have successfully expressed simultaneously 6 human proteins from genomic insertions using the following combinations: ProPGK1+terTDH1; ProTDH3+TerADH1; ProHHF2+TerSSA1; ProCCW12+TerENO1; ProTEF1+TerENO2; ProTEF2+TerPGK1. The most crucial point being to optimize sequences for expression in yeast.

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.

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 Siddhi Sudev

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 :)

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.