Molecular Biology - Science topic

If you are using Cy5 as an internal control fluorophore, you can choose another one in the same channel. An alternative is Quasar 670. Also, check your per-analytical procedures and confirm your cycling conditions if they are set as suggested by the kit. I hope this helps.

Hello sir, Asif Bilal

Just saw your response. It means a lot to me. Thank you so much for your time.

If possible, could you please forward the survey to other amazing scientists, It would help me a lot.

Any dource to get the E1a and E1b sequence from HEK293T cells

Dear Ioana-Dana Ganta, Asif Bilal, Fakhira Afzal, thank you very much for your answers!

The above RG discussions are related and may be helpful. Good luck.

The question of the structure of DNA is not a question of the origin of the Universe. It must and can be solved experimentally. But for this you need to have a desire to know the truth. The problem is lack of desire.

I think that maybe you have used blunt end digestion endonucleases, this makes inserts to be ligable in both directions (as your experimental results suggest). If that's the case, maybe this information is useful:

Both the Costa and Weiner or Delphi genetics Staby methods could fix this orientation problem.

Hi,

The supplier claims that their "sample release reagent" (nucleic acid release technology), can lyse pathogens at room temperature very fast, with no need to heating, centrifuging or replacing tubes, the sample DNA/RNA can be extracted quickly through simple operations. The reagent is applied for the pretreatment of nucleic acid molecules, to release them from specimens, then the released nucleic acids can be used for clinical in vitro diagnosis or for the detection through appropriate apparatus.

I agree with Kyle Skalenko that experimentation is needed but first contact the company to sort out whether they work in enzyme units or Kunitz units to define the activity of your rnaseA, Then assume that the activity drops by a factor of 2 for every 10c drop in temperature to get an approximation of how much enzyme or increased time will be needed

Concerning cancer research, Journal of Oncology and International Journal of Molecular Science provide good possibilities for publication of innovative works.

Gerald Rukundo Nivedita Sharma Olukayode Temitope Egbon thank you all.

Hi kamariah, thank you for sharing this.

I wanted to try this myself. Can you share the ratio between the DF4 and ug of plasmid that you used? I was thinking of trying 2,5ul of DF4 to 1,25ug of plasmid for 1 well of a 24-w plate, containing 300.000 cells. Is this in line with what you did?

Thank you very much!

Hello Bidisha Mondal

DNA stored in water for 16 years at –20ºC remained intact, but showed varying degrees of degradation when stored at 2–8ºC. Please see Fig 1 & 2 in the link below.

Good Luck!

There are two mechanisms responsible for proper transcript termination: in E.coli intrinsic termination and Rho-dependent termination. The Intrinsic termination is mediated by signals directly encoded within the DNA template and nascent RNA, whereas Rho-dependent termination relies upon the adenosine triphosphate-dependent RNA translocase Rho, which binds nascent RNA transcripts and dissociates the elongation complex. Although significant progress has been made in understanding these pathways, fundamental details remain undetermined. Among those that remain unresolved are the existence of an inactivated intermediate in the intrinsic termination pathway, the role of Rho–RNAP interactions in Rho-dependent termination, and the mechanisms by which accessory factors and nucleoid-associated proteins affect termination as reported on Annual Review of Biochemistry about six years ago

Dear all,

Did someone figure out the sequence of the whole plasmid? I need to express Addgene: pBF_HsPepT1 in Xenopus oocytes and would be grateful to get some information on optimal linearisation of the construct for IVT. I am also interesting in knowing whether the backbone harbours a polyA.

Any help in this regard would be highly appreciated.

Thanks!

Hello Chris Lee

You may introduce protective groups that will help prevent enzymatic degradation of the foreign protein.

Cyclization of foreign protein can increase its half-life by protecting it from the digestion of exo- and endopeptidases. Cyclization of peptides and proteins generate more rigid conformations, and it exhibits decreased susceptibility to enzymatic degradation.

You can also protect the foreign protein from proteases by covalently attaching it to a biocompatible polymer, leading to reduced immunogenicity, enhanced bioavailability, and enriched pharmacokinetic profile.

For more information, you may refer to the attached paper.

Best.

Use Carbonate bicarbonate buffer pH 9.6

Composed of:

1.59gm Na carbonate

2.9gm Na bicarbonate

In 1000ml DDW

addrd 1:1 to the diluted virus and incubate it at 4°C overnight and then washed

@ Subhash C. Juneja , @ Var St. Jeor , @ Sreyasi Das Many thanks for your suggestions and comments.

I will replace it with the DPX, which has fewer problems and pay more attention to the dehydration step.

Best regards.

Muhammad Abrar Yousaf do you want to completely shutdown the gene or you just want to insert point mutation, in which such gene might/might not be functional?? in case of knockout, CRISPR would be great. However, for silent/synonymous mutation, wouldn't "site directed mutagenesis" work fine? using CRISPR for single base sub, check this papers: https://www.nature.com/articles/s41598-019-41121-4

Thank you so much for your replies. Jack Andrew Connolly, I will insert this in between ybhD and ybhH, as done in this paper. .

Cheers!

hello everyone,

how could I calculate the results of my patients using Qiagen Multi-Analyte Elisarray Cytokine kit?

I will try sir.

Because this situation is in the tight-binding regime, you should use the tight-binding equation for the equilibrium calculation.

fraction of enzyme with ligand bound=

[(K_d + R_t +L_t) - square root((K_d + P_t +L_t)^2 - 4R_tL_t)]/(2R_t)

where R_t is the receptor protein concentration and L_t is the ligand concentration (^2 means squared)

For example, if K_d=0.168 µM, R_t=14 µM and L_t=28 µM, the fraction of occupied receptor is 0.988.

Hi

See the publication attached (Varkonyi-Gasic et al., Plant Methods, 2007). It explains the procedure with several examples, and uses a 'pulse RT procedure' using a 16 degree step., and it works. I have followed this approach for some miRNA analysis. See if it works for your piRNAs also

All the best

You have really put up a very important current issue as large number of existing highly reputed journals in the field of animal science have now been shifted to open access mode and so called paid journals. In addition, large numbers of new open access journals are emerging rapidly with APC. Its really challenging for low income and middle income countries scientists to find out the source of funding for publishing their high quality science in internationally reputed journals. No need to mention, but several highly reputed international journals are now charging in the name of making published article full PDF available online/download and to increase its accessibility/visibility globally.

However, scientists from major part of the world (except few countries where fund is allocated/available for research publications or the member of some or other organizations) are having similar issues that have resulted them to keep on searching appropriate highly reputed international journals (with high impact) that do not charge for publication (no publication charges) or do not have APC.

Coming to your point, although not all journals but still several journals in the field of animal science from major publishers like Elsevier, Springer nature, Wiley Online Library etc are accepting manuscript without any charges/APC.

Although I am not giving you the name of the journals herewith, you can go through these publishers website and I am sure you can find out several highly reputed internationals journals in the field of animal science for your research publications.

Go through the google search by adding "list of animal science journals with impact factor" .

You get a list then see the journals which are not showing open lock (indicative of open access, most of the open access journals are paid except few).

Search the journals that deals with the field of your research interest and see their impact factor. Once you are satisfied that they do not charge and having international reputation and indexing, available on major search engine like PubMed/PubMed Central etc., submit your piece of research article for publication.

There are several journals that give option of open choice: For examples, The Journal of Assisted Reproduction and Genetics publishes cellular, molecular, genetic, and epigenetic discoveries advancing our understanding of the biology and underlying mechanisms from gametogenesis to offspring health.

Authors who choose to publish open access in Journal of Assisted Reproduction and Genetics are required to pay an article-processing charge.

Best

Hi Clara Sánchez-Blanco

You used 10% gel and you say you had good results letting the dye get to the bottom of the gel.

How many amps did you use for gel run and membrane transfer?

How much time did you spend on these two steps?

The best way to do this is to prepare a sample of parasite DNA from a known number of parasites, make a dilution series and then use this as a standard curve. If you cannot obtain such a sample, there are ways round the problem. I work with pathogens that cannot be cultivated in cell-free media and which are difficult or impossible to enumerate. To prepare my standards, I use a plasmid containing the target sequence (several suppliers can produce such plasmids using a synthetic sequence; I use Genscript). DNA concentration can be converted to number of copies by dividing by the mass of the plasmid. Then you make a dilution for use in the standard curve.

The problem with this approach is that you have to know (a) the number of copies of the target sequence in the parasite genome and (b) the number of cells in a parasite. If you don't know this, and you cannot get an accurately counted sample of parasites, then absolute quantitation is essentially impossible.

Can I also get recommendations for universities offering masters in the same subject abroad as well. I am also looking for those options as well.

I think it will be best if you set up a couple of controls for that

1. you can set a tube with only water and no template DNA.

2. while setting up the cDNA synthesis protocol, you can have an additional tube where you do not add the Reverse transcriptase enzyme. This tube will give you signal from background genomic DNA if you have any.

Hope this helps

Umar Abdulmutalib Thank you

Hi,

There are plenty of ways to do so, but the relative complexity of the experiment depends upon your experimental system. Are you using a well-established experimental cell line, and is the gene expression constitutive and high-level? Endogenous expression or an engineered strain under control of a promoter?

In the latter cases you could attempt to fractionate your cells for example by density gradient centrifugation and then a Western blot against a tagged target, but this would require a reasonably high expression of a fusion-tagged locus. Otherwise, if your target has an enzymatic activity (or a particular affinity), you could monitor the activity of fractions or attempt target pull-down with a known ligand/interaction parter from each of them. Along with proper controls, this could validate that your protein is indeed trafficked from the cytoplasm to the nucleus as you say and would not necessarily require a target-specific antibody.

You can contact Barcode Biosciences, Bangalore.

How you make graphic design for Nature Journals (NPG). I mean which software you use. I want to publish in one of NPG journal

Dear Sogand Shayesteh

First, find the problem, the learning will be added to your mindset by default. Anyhow, youtube, Coursera or even some text (pdf) based tutorials are already available on the internet.

Dear Mrutyunjaya,

I am not really in a position to assess the likelihood of an RNA World scenario for the origin of life on Earth. However, I would like to point out that from an astrobiological standpoint, I think that we should keep an open mind on the huge variety and broad range of potential (evolutionary) pathways leading to life. Moreover, a lot also depends on the precise definition of "life" and from which stage or time onwards we classify certain phenomena as "life". If life exists outside of Earth, it may look very different to what we have become accustomed to on our "pale blue dot". Once we have confirmed at least a second, independent instance of a living system in the universe, we may also see clearer on how probable a RNA World scenario may have been for the case of Earth (even if we may never be able to reconstruct and verify the exact pathway...).

Thanks & all the best,

Julius

The following RG link is also very useful:

Kindly Visit..

Probably it is quicker if you email to the authors listed in the papers (you listed above) directly, and ask for some samples.

Also these are good:

1- Writing Research Papers. Student's Book: From Essay to Research Paper

Dorothy E. Zemach, Daniel Broudy, Chris Valvona - 2013 - 128 pages

2- English for Writing Research Papers (English for Academic Research)

Part of: English for Academic Research (11 Books) | by Adrian Wallwork | Mar 3, 2016

3- https://www.scribbr.com/category/research-paper/

EBV circular DNA benefit establishing latent infection and long _term persistance for viral genome.the also the circular DNA infected cells may well be ignored by the immune system.the attached ref.illustrate all the request:

Annual Rview of Virology.Vol.3:359_372

Epsten_Barr virus is another herpes simplex 1

Also check https://www.ibiology.org/talks/fluorescent-probes/

Definitely a fair assessment, Ive made other samples that have similar properties as this, but none of the other samples Ive generated have responded with as much vigor as this sample.

Definitely will invest in generating another panel of T-cells but from what I can tell so far, this sample had a particularly rare phenotype that i may or may not see again in a relatively limited panel size. every once in a while I remember I have this last vial and wonder if I can do anything with it, in the end its always for the birds...

sorry

outside my area of expertise

ORFs of eukaryotes are typically different from those of prokaryotes. In eukaryotes, mostly, genes do not come as long ORFs; they are "mixed up" with introns. Thus, providing substantially large spaces for non-coding genes (which has been demonstrated to be as high as 62% in the human genome). In bacteria, there is relatively very low non-coding DNA in the genome (approx 11%) which therefore gives in a continuous ORFs. What may be located between ORF's of two different genes, or the intergenic regions may comprise of the list succinctly put by Dr Frank Burns. It is also however, important to remember that some overlaps do occur that complicates the assessment in a single shot.

It probably depends upon the enzyme but you might get some cleavage however it is not likely to be efficient. The reason you need two is the enzyme must dimerize in order to cleave and you only get one monomer per recognition site.

Sam Augustine Kandathil NO sir, I didn't used coated plates

In that case Jessica Little the handbook on that page suggests 10exp8 cells should produce between 25 and 70ug RNA depending on the growth medium used so it looks like you can count cells,spin them down,decant most of the supenatant and then make up to a small convenient volume and add rnaprotect. You might contact the company making the rna for you and check with their tech support how and in what volume they want their samples sent. They will have a lot of expertise in converting various sample types and volumes to RNA

Not only ligand concentration but pH is also an important factor of chelation .

I understand. That makes sense. Thanks for the clarification. This is very helpful!

Aerobic glycolysis creates an acidic micro environment that compromises the TEJ of the normal native vasculature creating vasogenic edema which increases the glucose concentration in the tumor micro environment, favoring unregulated cellular proliferation. MREPT can detect vasogenic edema enabling minimally invasive ablation of cancer before cellular mass inhibits diffusion, and much before the formation of tumor vasculature can create “washout “.

Hi Kimberly A Wong , thanks for sharing this. I am used to working with The Step One Plus software and exporting files to LinReg, but I have recently started a short research stay at another lab and they have a BioRad qPCR machine, they do not use LinReg to analyze their data, so I am struggling with this. Do you know if there is a preferential order to put the samples on the plate so that the resulting data is more organized and easier to read after processing with Linreg? I ask this because with Step one plus it is simpler if for example, you pipette the technical replicates in continuous wells in the same row.

Thank you

يمكنك مراجعة اهل الاختصاص

Kindly check the following RG link that discusses commonly used techniques for the detection and diagnosis of viruses in clinical samples.

The non-uniformity of shingles occurrence does not violate Koch's first postulate since all patients with shingles exhibit active VZV. The converse (All VZV infections must cause shingles) is not necessarily required by the first postulate or even the third postulate. You can find several good examples of why the postulates still hold true for shingles/VZV in the paragraph below the list on that Wikipedia page you linked.

Causality can be established using several tests for VZV during a shingles episode including checking for anti-VZV antibodies from blood and PCR for VZV directly from shingles blisters. (https://en.wikipedia.org/wiki/Shingles#Diagnosis)

Dear Clarence,

The most obvious advantage is the larger genome. Unlike RNA polymerases (I mention the majority of the enzymes excluding the polymerases of the order Nidovirales), DNA polymerases have proofreading activity. Consequently, the mutation rate for DNA viruses is at least 10-100 times lower than for RNA viruses. For RNA viruses, it is almost impossible to construct the genome exceeding 10 000 nucleotides. That means that RNA viruses are usually limited by 10 proteins (it is not so simple to override cellular defensive mechanisms, orchestrate your own reproduction and escape the immune system if you only have a several, or even a couple, of non-structural proteins). Double-stranded DNA viruses usually have about 100 000 nucleotides genomes, and some of them have reached even more that 2 million nucleotides (Pandoraviruses). The larger genome allows encoding the larger number of non-structural proteins. For example, herpesviruses, the typical double-stranded DNA viruses, encode from 70 to 200 proteins.

Leonid Volkov and Takayuki Kitagawa here is a recent and well illustrated review on G6P exchange and G6Pase in endoplasmic reticulum

Hi!

I obtained this protocol provided by a company that sells that kit.

Purify DNA from cell or tissue samples by a desired method or commercial DNA purification kit. Reagents needed but not supplied: Nuclease P1 3M Sodium Acetate, pH 5.2 1M Tris pH 7.5 Alkaline Phosphatase Zinc Chloride For digestion, 15 µg of DNA in 100µL DNA hydration buffer or DI water is required. Protocol volumes can be scaled. 1. Prepare working solution of Nuclease P1 at 5U/mL in 40mM sodium acetate. Keep on ice. Remove aliquot of alkaline phosphatase, 10U/mL, from -20˚C. Keep on ice. Thaw normalized DNA samples (15 µg/100 µL). 2. Denature the DNA at 95-100˚C for 10 min. Cool completely on ice 5 min. Centrifuge for 5 sec or tap any condensate down into tube. Add 50 µL 40 mM sodium acetate pH 5.0-5.4, 0.4 mM ZnCl2 . 3. Add 50 µL of 5U/mL Nuclease P1. Invert tube to mix. Centrifuge 5 seconds or tap any condensate down into tube. Incubate at 37˚C for 30 min. 4. Adjust pH to 7.5-8.0 by adding 20 µL 1M Tris pH 7.5 to tube. Add 15 µL of 10U/mL alkaline phosphatase. Invert to mix. Centrifuge 5 sec or tap any condensate down into tube. 5. Incubate at 37˚C for 30 min. Boil samples for 10 min at 95˚C to inactivate alkaline phosphatase. Place samples on ice. Aliquot samples, 2 µg/tube and store at ≤ -20˚C until assaying. Samples should be diluted ≥ 1:4 with the diluted Assay Buffer prior to running in the assay.

Hope this helps!

Dear Dr. Zonghan Gan

I think your answer is here

The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K(d), in the order of 4x10(-14) M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. That a short incubation in nonionic aqueous solutions at temperatures above 70 degrees C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology.

nuha hamid taher

Senior lecturer

Faculty of Basic Education

Mustansiriya University

Whereas It is possible

Red biotechnology: This area includes medical procedures such as utilizing organisms for the production of novel drugs or employing stem cells to replace/regenerate injured tissues and possibly regenerate whole organs. It could simply be called medical biotechnology.

nuha hamid taher

Senior lecturer

Faculty of Basic Education

Mustansiriya University

The change in the level of uric acid and biochemical parameters in a patient with an identified case of desminopathy is presented in the article https://www.researchgate.net/publication/357311034_CHANGE_IN_REDOX_STATUS_AND_BIOCHEMICAL_PARAMETERS_IN_PATIENT_WITH_DESMINOPATHY_T341P_SEVERAL_YEARS_AFTER_DISEASE_SYMPTOMS_ONSET

Electroporation has been used successfully to deliver plasmid DNA to a variety of tissues in vivo. Because of its physical nature, EP can be applied to practically any cell or tissue. Plasmid DNA in the appropriate diluent is injected into the tissue. Electrodes are then placed around the injection site and the cells within the tissue are subjected to a high-voltage electrical pulse of defined magnitude and length. The animals are then allowed to recover and the tissue is evaluated at specified time points following delivery. Factors that can be varied to optimize electroporation effectiveness are pulse width, number, amplitude and electrode configuration.

autoradiographic and pharmacologic technique

Because in 1992, Okayama et al. demonstrated the presence of H1 receptors in the mucosa of human turbinates by using autoradiographic and pharmacologic technique.

The answer is found in this study:

J Virol. 2006 Nov; 80(22): 11124–11140.

Published online 2006 Sep 6. doi: 10.1128/JVI.01076-06

PMCID: PMC1642140

PMID: 16956935

Recombination and Selection in the Evolution of Picornaviruses and Other Mammalian Positive-Stranded RNA Viruses

If you pursue that medical science route, chances are that you would be pursuing a terminal degree (PhD) and doing research in academic setting.

Unless you really love and good at biochemistry/molecular biology, I personally discourage it. I would encourage you to consider pursuing a degree in bioinformatics. Your skill sets would be highly valuable in many different fields, including chemistry and molecular biology.

Thanks a lot for your answer and suggestion professor Iman Hassan Ibrahim and Dr Abhijeet Singh

You can ,as you say, pcr amplify overlapping 800 base amplimers ( you get no useful sequence under the sequencing primer or for the next 30 bases). If you can amplify the whole 6KB in one amplimer then you just need overlapping sequencing primers at 800 base intervals along the whole sequence but just the one template dna. If you have a friend with a minion sequencer or NGS then new sequencing technology is good and quick but not cheap or easy to analyse the results

The polymerase does not know that. The thing is that, in the first step, the polymerase will copy everything (into a complementary strand) from the primers. In the second step, this complementary strands now will have attached the another primer, so it delimits the region to be copied. Let's put an example focusing only on one strand: the direct primer will allow the polymerase to start the amplification from an end. After this step, you will have already delimited one part of your amplicon. In the second step, as the strand is the complementary one now, the primer bound will be the reverse, so delimitating the another end of your amplicon.

I don't know if I was able to explain myself as it is difficult to explain by words, but I hope it helped you

Which cells do you want to reach ?

It is extremely important to know the target location. e.g. blood-brain barrier.. 1-2 dosages are sufficient, intracerebral (e.g. astrocytes, neuron) can take a while.

Depending on your animal facility you will have difficulties due to quarantine time

of the tamoxifen.

A biotinylated primer can be used under the same reaction conditions that would be used in an unmodified oligo. If you do have trouble getting an efficient reaction, a Mg2+ and/or annealing temperature optimization process can lead to more efficient amplification.

Check https://www.jbc.org/article/S0021-9258(21)00607-4/pdf

Should work, but try it at an inconspicuous spot first.

SDS should be fine, too, as it doesn't stick to DNA. It is used together with NaOH for lysis and removal of proteins and (protein associated) genomic DNA in plasmid isolation.

Also check https://www.promega.com/resources/pubhub/head-to-head-comparison-of-rnasin-to-rnaseout/