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Molecular Biology - Science topic

PCR, Cloning, Restriction Digestion, Ligation, Transformation, Plasmid et al
Questions related to Molecular Biology
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Hello Everyone,
Recently when I am working with GMO by RT-PCR using r-Biopharm kit, I didnot get the NOS results in CY5 channel even in positive control. Even though I repeated twice, I did not get the NOS. So, I felt there might be some issue with CY5 channel in our RT-PCR. In this regard, I would like to request you all that are there any other alternative channel for CY5 or can some one please clarify why I am not getting NOS in CY5?
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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.
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I am a student of biotechnology and an independent SARS CoV-2 researcher from India. For finding the academia research status and analysis of the integration of innovation with research, I have created a set of Multiple choice type questions about your experience as a researcher. The google form requires nothing but your honesty and openness for research. Feel free to ask questions and DM. The questions will assist in gauging the level of innovation and writing in academia.
If possible, please do forward this little form to your fellow researchers and other amazing scientists. I would be highly grateful.
Thank you
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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.
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I would like to do PCR-RFLP for specific SNPs detection that's why i need to design primer
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Any dource to get the E1a and E1b sequence from HEK293T cells
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To save life in desminopathy, can the body purposefully reduce muscle mass, for example, due to decreased heart function or for another reason?
It is known that when hypothermia, the body sacrifices limbs for survival. Is it possible with desminopathy a similar phenomenon?
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Dear Ioana-Dana Ganta, Asif Bilal, Fakhira Afzal, thank you very much for your answers!
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I am working on two SNPs on the same gene, and I tested some biochemical parameters for 150 patients with hypothyroidism. I want to see if a certain haplotype has an impact on these biochemical parameters. How can I statistically calculate the haplotypes and their association with these parameters?
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Does the DNA remain stable or degrade at this temperature? Would there be any difference in thermal stability between supercoiled and linear forms of say, 3 kb plasmid.
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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.
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Hello everyone,
I did the assembly of chloroplast genomes for some Boraginaceae species, in a few species, I got an orientation problem where the rbcl gene position is within the circular shape in a clockwise direction, and the atpB and atpE genes position is are outside the circular shape in the anti-clockwise direction (please see the picture), and this is a different result from most assemblies of chloroplast genomes that have been published !!!. (usually, the rbcl gene is outside the circular shape and in the anti-clockwise direction while the atpB and atpE genes are within the circular shape and in a clockwise direction)
I am using Chlorobox to draw the gene map after Novoplast finishes the assembly, this issue happened with only two of 7 samples, the two samples are from the same family !!!, I change the seed and reference many times and still got the same result.
Any idea what I need to fix this?
Thanks!
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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.
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Hi,
I wonder if anyone knows how to use sample release reagent from Sansure Biotech ?
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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.
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Long story short, I need to degrade 30ug of RNA and i need to do it at 4C. I want to use only as much RNAse A as necessary.
So if performing the reaction at 4C, how long of incubation time and how much RNAse A would i need to degrade 30ug of RNA?
For example, would 1ug of RNAse A(20ug/ml conc.) for 15min at 4C be enough?
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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
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My research is about micro RNA in breast cancer and I have to publish the research in an SCI or extended SCI journal. Would you please suggest journals that can response in real-time with logical prices?
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Concerning cancer research, Journal of Oncology and International Journal of Molecular Science provide good possibilities for publication of innovative works.
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Hi all,
Bubbles affects the absorbance reading from my assay. How can avoid bubbles when pipetting with multichannel pipette.
Thank you
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  1. Maintain consistent pressure: Use consistent pressure when you’re pipetting, and when you’re assembling the pipette. When you attach your pipette tip, make sure to avoid a fit that’s too loose or too tight. Use consistent force to apply your pipette tip to avoid fit issues that might affect your measurements.
  2. Focus on angles: To ensure you dispense all the liquid in your popette and avoid air bubbles, aspirate at a 90 degree angle and dispense at a 45 degree angle.
  3. Release pipettes slowly: After dispensing the liquid in your pipette, you shouldn’t release the plunger too quickly. Letting go of the plunger suddenly may cause air bubbles that can affect liquid measurements in your pipette. Release your plunger slowly with a controlled motion after you’re done dispensing.
  4. After using your pipette, flush residual substances out by aspirating with water and dispensing it again. Flush your pipette out two to three times before using it for your next sample.
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On the molecular biology level, what is the authoritative method to distinguish Lactobacillus casei and paracasei (or whatever they are called now) ?
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Dharmafect 4 (DF4) from Dharmacon is commonly used to transfect siRNA. However, I have trouble with my Neon transfection to transfect my plasmid. I accidently tried using DF4 to transfect plasmid into my cells and got successful results. But, I am a bit worried as a lot of publication use DFs are commonly use for sirna. Kindly anyone please advice.
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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!
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As we have started molecular biology programme in UG, we want to make it low cost
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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!
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It is about Molecular Biology. Please answer up. Thanks
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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
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I looked up in the internet but couldnt find the antibiotic resistance. Someone knows? seems to be an oocyte expression vector...
Thanks!
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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!
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If I wanted to introduce a foreign protein into the human stomach without it getting cleaved up by proteases such as trypsin, how would I do so?
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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.
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Hello everyone,
I hope this message finds you doing well.
​I am writing to ask you a significant question about whole-virus ELISA and its procedure.
Honestly, it seems that coating a microplate with viruses is not as convenient as some papers mentioned, especially when high accuracy is needed. Now, my question is, is there any particular procedure in order to enhance the efficiency of the coating? For instance, what would we do if we tended to expose viral protein to the microplate better than before, based on your experience?
Thank you
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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
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I am wondering if anyone has an explanation for the appearance of these clumps on IHC stained sections following the use of Canada balsam as a mountant! Any ideas to get rid of them?
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@ 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.
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I am interested in evaluating the role of silent/synonymous mutation using the CRISPR-Cas9. However, I am unable to find any literature related to it. Can you please provide data related to it?
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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
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I am planning to use the Lambda Red recombineering system to insert an insertion in the following format depicted in the picture to E.Coli genome.
My questions are
1. Is the T7 promoter suitable for this purpose ? If not what promoters are better ?
2. Can I include the lac operator also in the insertion?
I really appreciate any guidance from you.
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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!
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By using this cytokine kit I am getting high absorbance value in my positive controls. Recommended values for positive controls are 1.5-2.5, but I am getting values around 3.0-3.5.
In some of my negative control I have absorbance more than the experimental sample. Eg. negative control value 0.04 and sample value 0.01.
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hello everyone,
how could I calculate the results of my patients using Qiagen Multi-Analyte Elisarray Cytokine kit?
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On this eve of mahashivratri, I would like to share my opinion that, Soon quantum computations explore that, ingenral, bipartite graphs are not symmetric about the origin.
This may may lead new directions in the study of quantum mechanics, molecular biology in particularly genetic engineering.
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I will try sir.
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The concentration of my protein is 14 μM and the Kd is 168 nM. I want to have the ligand in excess, but I am not sure how to go about it.
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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=
[(Kd + Rt +Lt) - square root((Kd + Pt +Lt)^2 - 4RtLt)]/(2Rt)
where Rt is the receptor protein concentration and Lt is the ligand concentration (^2 means squared)
For example, if Kd=0.168 µM, Rt=14 µM and Lt=28 µM, the fraction of occupied receptor is 0.988.
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I am working on measuring piRNAs using stemloop primers. I am kind of confused about their designing process, though. Do I purchase the oligo DNA primes? Will the primers form the stemloop during the PCR process? Or do they need to be folded first?
I will be using sybr green qRT-PCR.
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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
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I need a list or site where I can easily find journals with no APC. The journals' scope should be Animal sciences, Biotechnology, Genetics and Molecular Biology.
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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
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Does anybody have experience with ACC (Acetyl Coenzyme A Carboxylase) and pACC detection by western blotting?  It is often used to confirm the status of AMPK activity. The antibodies we use are from Cell Signaling Tech. and they are cited in several publications. However, our anti-ACC does not work at all, while anti-pACC recognizes an unspecific sharp band at 100 kDa and a signal, which  is running around the expected MW 265 kDa,  not as a defined band, but as a “diffused zone”. It should be pACC, because its intensity mirrors that of pAMPK. I am not sure, if it is a problem related to our SDS-PAGE 6% gels or blotting or lysate preparation (RIPA buffer + protease and phosphatase inhibitors, then treated 5 min at 95 °C in highly reducing  conditions (beta-mercaptoethanol, 5% in lysates). We are trying to detect ACC in HepG2 cells and rat liver lysates. This is really frustrating, because according to several publications pACC and ACC should be sharp bands. Please help us to identify the critical problem and get nice pACC/ACC blots.
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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?
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While Working in molecular biology Lab, QuantStudio™ 3 Real-Time PCR System, 96-well, 0.2 mL, laptop is our tool.
I want to calculate the Genomic equivalence, then want to calculate it for parasite number per micro liter of blood.
Hence, I am looking advises from any one working on this area is very helpful.
Abdissa B.
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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.
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i am in my 6th semester and i am looking for some good colleges other than IIT's that are offering masters in immunology and molecular biology in india. can i get some recommendations on the same?
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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.
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Hello everyone,
Could somebody tell me about, how to interpret the Zero or No expression of target gene over the control population.
"Whether getting zero in one population and more than one in another population of the same of the similar can be acceptable?"
Your reply appreciated!
Thank you.
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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
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I want to know which institute's in India offer synthetic biology courses>?
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I want the name of techniques which assist in the analysing the translocation of protein, apart of fluorescence microscopy, TIRF or Fluorescence immunostaining. Which technique is best one for studying translocation of proteins..
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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.
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We would like to purchase around 10 thousand DNA oligos in a 96 well format (25 nmol). The cost per base is coming to around Rs 14-15. We wonder if there is any economical option available in the market.
Thank you
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You can contact Barcode Biosciences, Bangalore.
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Researchers in cellular and molecular biology who have difficulty publishing articles can contact me for guidance.
I am ready to share my experiences in publishing articles.
Please add in the subject section: Cellular and molecular biology
E. mail:
dkahrizi (at)yahoo.com
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How you make graphic design for Nature Journals (NPG). I mean which software you use. I want to publish in one of NPG journal
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Hi all,
I want to learn how to work with these databases? I do not know how to learn them step by step, and there is no instructional video.
-Genevestigator
- ProteoCloud
-PeptideAtlas
- Chorus
Thanks for all your help.
#molecular_biology
#proteomics
#functional_genomics
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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.
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Kindly discuss your ideas and viewpoints on the origin of life and the RNA world hypothesis.
What are the contradictory views on why researchers are still unsure about the origin of life through RNA or such analogous molecular intermediate pre-cursors preceding its existence?
"The general notion of an “RNA World” is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA and genetically encoded proteins were not involved as catalysts. There is now strong evidence indicating that an RNA World did indeed exist before DNA- and protein-based life. However, arguments regarding whether life on Earth began with RNA are more tenuous. It might be imagined that all of the components of RNA were available in some prebiotic pool and that these components assembled into replicating, evolving polynucleotides without the prior existence of any evolved macromolecules. A thorough consideration of this “RNA-first” view of the origin of life must reconcile concerns regarding the intractable mixtures that are obtained in experiments designed to simulate the chemistry of the primitive Earth. Perhaps these concerns will eventually be resolved, and recent experimental findings provide some reason for optimism. However, the problem of the origin of the RNA World is far from being solved, and it is fruitful to consider the alternative possibility that RNA was preceded by some other replicating, evolving molecule, just as DNA and proteins were preceded by RNA." - Robertson and Joyce
[This is as per the explanation by Michael P Robertson and Gerald F Joyce in the article: "The origins of the RNA world." published in the Cold Spring Harb. Perspect. Biol. 4, a003608 (2012).]
The scientific community must resolve this contradicting conjecture through rational discussion and debate backed by strong experimental evidence on what must be the pre-cursor molecule to the Origin of Life if it is not RNA!
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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
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Plaques form due to a self-healing mechanism of blood vessels and will increase over time. When entering blood vessels, they block blood flow, lead to hypertension and decrease blood flow to organs such as the heart. To get rid of these plaques, we need to boost the good cholesterol such as HDL or improve health of liver to produce enzymes that move these plaques. So, what other ways to get rid of these plaques without using invasive methods?
Thanks and best regards.
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The following RG link is also very useful:
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How could you judge someone by publication and impact factor of journal or publisher?
What you think it’s easy to publish work in a high impact factor journal without funding? Nature and MDPI and Hindwai and PlosOne and other good journals are open access...and having Article processing charges...
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Hello everyone,
I'm interested in a protein localised in apoplast. To confirm, I want to create fusion with a fluorescent protein which would be stable in acidic environment. Based on literature I selected the following:
cyan
mTagBFP2 - Subach et al. 2011
(mCerulean3 - Markwardt et al. 2011)
red
mCherry2 - Shen et al. 2017
TagRFP675 - Piatkevich et al. 2013
FusionRed - Shemiakina et al. 2012
Would anybody be willing to share them?
Thank you
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Probably it is quicker if you email to the authors listed in the papers (you listed above) directly, and ask for some samples.
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I am trying to check expression of hsp-70 by reverse transcriptase PCR in human sperm. But I am not getting anything. However I checked the quality of cDNA by internal control (GAPDH expression), and the cDNA was fine. When I use the same primers for other sperm/somatic cell samples, the primers are working fine.
Can someone please suggest how to overcome this problem?
Thanks
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Before and after exercises would be an amazing topic to search
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We are trying to build up a "Quick Step Guide for Young Scientists Playlist" to assist young researchers in their journey of scientific writing and publishing. Playlist link: https://www.youtube.com/playlist?list=PLwyWKnsS4EkgcQ1Wnkx7FIxUsLsLUp0gb
We hope it will help many of you.
Share it with your friends and colleagues. All the very best!! Channel link: https://bit.ly/Subscribe_Learn_SciTech
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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
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How does circular DNA benefit Epstein-Barr virus (EBV)?
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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
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Our quantum dot(g-C3N4/CQDs) was prepared from melamine and then exposed to phenylbronic acid(PBA) for the next steps of the experiment - whose fluorescent properties were quenched (Figure 1) but after 5 days and quantum dot solution dialysis with PBA (1000D) A little of its fluorescent property back, what is your analysis?
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Hello!
I isolated some T-cells with some very interesting TCRs from primary cultures. I sent them to Genewiz to get chromium single cell TCR sequencing done, however the sample viability was super low. I sent them 8 more vials so that they could do a dead cell removal and then isolate the live population and perform single cell sequencing on the remaining cells. The sequencing results show that whatever is left over after DCR is most likely another contaminant cell type, not a TCR. I now only have one vial left, so whatever I choose to do next is very critical and essentially has to work the first try.
At this point I dont care about chain pairing, I can piece that back together afterwards by trial and error, I just want to get some data from these cells. I was wondering if anyone knows if I can thaw my cells directly into RNA later and then do either normal NGS or another single cell sequencing method to get any info on the TCR sequences? Should I just amplify the TCR regions on thawing with some kind of primer pool and then send that for NGS? In general, what's the most robust process for getting out TCR information from low viability samples?
Some other notes:
1. I didnt personally do the T cell isolation but my thinking is they were pretty much exhausted at the time of freezing which is why we have viability issues on thawing
2. They were frozen in 10% glycerol + 10% FBS in a Mr Frosty at -80C and then maintained in LN2 and shipped on dry ice.
3. Observed viability is ~30% on thawing however this could just be the contaminant cell population....
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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...
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"The development and validation of a medium density SNP genotyping assay in Shrimp" is a research proposal I'm currently working on. Given the restricted budget allotted (9,600 USD) to the project, I'd like to know ahead of time how much it might probably cost me.
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sorry
outside my area of expertise
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I know that several genes come one after the other under a single promoter in an operon, but what is exactly between those genes? Does the start codon of the second ORF come right after the ORF of the first gene? If there is a specific example with the dna sequence, that would be great.
Also, does an operon always require an operator?
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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.
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Type IIS restriction enzymes such as BsgI typically cleave when two restriction sites are present, does anyone have any experience trying to use these enzymes when there is only one site present? Is it possible?
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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.
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in passage 0 the cells grown properly but after subculturing they did not adhere to the flask and showed cell death(round in shape and less number).Some of the cells that are frozen in -80 degree was revived in T75 flask the load found to be too low and no proper cell growth, cells are found to be round in shape and floating. What may be the reason? Kindly suggest me the possible reason for the above mentioned problem.
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Sam Augustine Kandathil NO sir, I didn't used coated plates
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Hi all,
After having unsuccessful RNA extractions doing it myself the first time, and having the impeding time pressure of final year of my PhD, I am looking to send off some bacterial samples to novogene to have the RNA extracted from them for RNA seq.
I want to use RNAProtect to stabilise the RNA so that minimal degradation occurs.
How do I use it? I have read that it is generally 2x volume of culture, but I am working with 100ml cultures... Would there be a way to minimise RNAprotect consumption? Eg. Could I pellet a 100ml culture, resuspend in 10ml, for example, and then only use 20ml RNAProtect?
Thank you in advance
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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
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Most people say that imidazole doesnt affect the majority of downstream applications for purified proteins but it is a chelator, so you would think that it might chelate the Mg2+ in solution and inhibit Mg2+ dependent reactions. Anyone seen any papers that discuss what concentration Imidazole will chelate magnesium or manganese ions in solution?
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Not only ligand concentration but pH is also an important factor of chelation .
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I want to do a toeprinting assay to map translation start sites. Typically, these experiments are carried out with radioactively labeled oligos that are extended using reverse transcriptase. However, working with radioactivity is difficult in my institution, and I would like to avoid it.
I am thinking of an alternative. Specifically, what I am planning to do is to use 5'-biotinylated primers, then employ reverse transcriptase as in the standard protocol, then run the primer extension products on a PAGE gel, then transfer it onto nitrocellulose membrane just as if it were a Western blot (I would use the exact same protocol, just leaving out the SDS), and finally stain the membrane with streptavidin-HRP.
To my surprise, I nowhere found a protocol like this in the literature. Instead everybody keeps working with radioactively labeled primers. This suggests to me that my plan is probably a bad idea, because somebody must have tried this, right?
If anyone would like to way in, I would appreciate an opinion. I'd be happy about any support for my experimental layout, any concerns why this might/will not work, or suggestions on how I could optimize the plan ...
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I understand. That makes sense. Thanks for the clarification. This is very helpful!
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Could someone kindly clarify why aerobic glycolysis is required for cancer cells? That is, if tumor cells used OXPHOS, would they be unable to grow and metastasize as well?
The most commonly cited benefit of aerobic glycolysis seems to be accelerated glucose uptake, but why is this required for long-term growth as opposed to short-term spurts? The timeline for cancer progression is normally months or years, not hours or days.
Moreover, despite consuming glucose faster, total ATP production from aerobic glycolysis is lower. That is, during the time OXPHOS consumes 1 glucose molecule, aerobic glycolysis may consume 13 glucose molecules -- yet this yields fewer ATP molecules.
If correct, total energy production inadequately explains the relationship between cancer cells and aerobic glycolysis.
Is the timing of ATP essential? Perhaps cancer cells need fewer shots of ATP molecules but at a higher frequency rather than wait for one large batch from OXPHOS?
Or what are the other benefits of aerobic glycolysis over OXPHOS?
Ultimately, why would cancer cells be less successful with OXPHOS?
Thanks in advance for your help.
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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 “.
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I use the BioRad CFX manager to analyze my qPCR data. I want to try to conduct an analysis of primer efficiencies using LinRegPCR, but when I try to load the information into LinRegPCR I get errors. I am not sure if it is because my data export is not in the appropriate file format for the LinReg program.
I have tried both exporting RDML files and it says "list index out of bounds (1)", which suggests the exported file is empty. Similarly, importing the data via Excel from the Quantification Data exported as a csv produces an error of "cannot convert variant of type (OleStr) into type (double).
Has anyone had success taking qPCR data from a Biorad machine into LinRegPCR (or any other R or MATLAB program)?
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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
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Asthma is a chronic, obstructive disease;
In asthma we have hypersecretion of mucos ;the main component of mucos is mucin ; the main airway mucins are muc 5ac and muc 5b that are released from goblet cell and submucosal glands ,respectively.
Asthma characterized by some changes, like: thickening of the lamina reticularis, epithelial shedding, subepithelial fibrosis, inflammatory cell infiltration, goblet cell hyperplasia, myofibroblast proliferation, smooth muscle hyperplasia and hypertrophy, and neovascularization of the airway wall
According to the findings ; increased amount of the muc5ac and decreased amount of muc5b is observed.
Goblet cell hyperplasia can cause more expression of muc5 ac but there is no evidence for the reason of decreased amounts of muc5b .I'm looking toward this decreasing reasons.
I will be thankful if you share your ideas with me .
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يمكنك مراجعة اهل الاختصاص
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I'm curious to learn more about virus detection.
What are reliable methods for detecting viruses inside a host?
If a virus integrates into the host genome, if given a viral sequence, is analyzing the host genome for matches reliable? In other words, is it impossible for viral genomes to change before attaching to the host genome?
If a virus doesn't integrate, is scanning for viral proteins sufficient? Presumably scanning for viral sequences is not possible as the viral genome is not exposed?
Put another way, what evasion mechanisms do viruses deploy to avoid detection?
Thanks in advance for your help!
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Kindly check the following RG link that discusses commonly used techniques for the detection and diagnosis of viruses in clinical samples.
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My understanding is that proving causation between pathogen and disease requires satisfying Koch's postulates (https://en.wikipedia.org/wiki/Koch%27s_postulates).
It is accepted that the varicella-zoster virus causes chicken pox and shingles, yet the shingles causal relationship violates the first postulate: not everyone with the virus develops shingles.
Could someone kindly explain how researchers proved the causal relationship between varicella-zoster and shingles?
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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)
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Particularly in the context of infecting humans, what advantages would DNA viruses have over RNA viruses or vice versa?
Is it accurate to say that DNA viruses are more more dangerous because larger genomes can encode more viral proteins, allowing for more countermeasures and redundancies against host defense systems while also producing more complex proteins capable of sophisticated exploitation of host cell metabolism?
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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.
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Dear all,
I'm currently interested in the hexosamine-pathway and especially in the enzyme Glutamine fructose-6-phosphate amidotransferase (GFAT).
The end product of the enzymatic reaction of GFAT is D-glucosamine 6-phosphate. It is known that glucosamine can enter the cells when added to cell culture via glucose transporters, but there is no data in the literature whether D-glucosamine 6-phosphate can be transported by these transporters as well. Does somebody know whether D-glucosamine 6-phosphate can enter the cell through glucose-transporters or maybe via alternative ways? And my second question is, whether D-glucosamine 6-phosphate is stable in cell culture conditions at all?
Kind regards,
Robert
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Leonid Volkov and Takayuki Kitagawa here is a recent and well illustrated review on G6P exchange and G6Pase in endoplasmic reticulum
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I need a concrete and already tested and approved protocol for DNA digestion using nuclease P1 and alkaline phosphatase. We're going to measure oxidation in DNA bases using a standard kit, but prior to the procedure we have to digest DNA into separate nucleosides. Have any of you applied such a protocol before? Can you recommend any protocols or articles to look into?
Does anyone know the amount of the nuclease P1 and Alkaline phosphatase I would need to add to digest DNA?
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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!
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Hi!
heating in formamide buffer is a common elution method to disassociate streptavidin. Yet is this disassociation irreversible when afterwards formamide is diluted and cooled?
Thanks!
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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
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I am aware that TiO columns are used to enrich phosphorylated peptides after digestion of proteins, but can they be used to purify full phosphorylated proteins from crude cell extracts? Something around 30 kDa
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Whereas It is possible
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The Fig2A of the paper shows that a tiling library of a gene was prepared containing 50bp fragments. The fragments span over the entire gene sequence incrementing about 7bp from each other. Later this sample was used to study the sequence dependence on DNA bendability over genome scale. This is a very interesting study but I am unable to figure out how the tiling library was prepared. Is it done by preparing a sequences for primer pool for every fragment and ordering them? Can we prepare a tiling library with any amount of spacing between them? Please let me know if you have any idea.
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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
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In a patient with hereditary desminopathy (mutation Thr341Pro DES in the heterozygous state) over the past three years, an increase in the blood uric acid level up to 440-480 µmol / l was established by 1.5 times (the norm is 428.4 µmol / l). With the progression of the disease, the level has risen and is above normal. It is known that uric acid is an antioxidant. Is it necessary to reduce the level of uric acid? The patient has no problems with the joints.
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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
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Hi All,
I am fairly new to PCR techniques (been at it for ~4 months now) and I have never posted here so if there is a problem with the amount or type of detail I provide (i.e., not enough, incorrect lingo, etc.), please offer suggestions to fix. Thank you.
Equipment: Cepheid Smart Cycler II
Gene: TRH
Avenues tried: triple-checked probe and primers, new probe w/new channel, probe developer consult (it passed all the checks on their end so there wasn't much they could do for us), various levels of probe in mastermix, different temp protocols, different dye settings (FTTC25 vs FCTC25), fresh mastermix components*
Results: the gene is amplified in our positive control when I run the gel, but stays negative for the smart cycler Ct.
The problem: For months, trh probe w/Tet Cy3 (new Feb 2021) was performing well on our smart cycler. A few months ago (June-ish 2021), the machine stopped giving us a signal. Even the positive control wasn't working. I tried to test it on a different machine but that ended up being a flop because of settings and no history of the machine reading that dye. Time was being wasted on that avenue. So, we went ahead and got a new probe with a new dye (FAM) that we knew our machine was detecting well. We're still getting no signal in our positive control...
Next: I am going to run our other FAM probe to rule out a mechanical issue. Maybe the channels are failing one at a time.
*mastermix is still working well for other genes that were being amplified simultaneously and for the internal control.
Does anyone have experience with smart cyclers, have had similar experiences, or have had their probes unexplainably not work? I will take any and all advice into consideration even if your situation is vaguely similar!
Thank you for reading,
M.
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  • This is probably one of the best questions in the sense it was asked, where you have put real efforts in explaining your problem with a lot of self troubleshooting before this question. Highly appreciated.
  • I have never used this machine so can't comment on this, but as you explained you have tried several things, but it did not work. I might not have a direct answer as well, but is it only this particular gene on this particular machine not working or nothing is working on this machine in a successful way? If I get it right, the other machine did not work because of settings. Could it be that you machine need to calibrate or need service? If you are getting signal in normal PCR/gel, then your machine have to have problem and maybe not the primers/probes.
  • Have you tried yur older primer/probes and/or new primer/probes on a normal qPCR machine?
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I want to reuse electroporation cuvettes for transformation of new Plasmids (different than one already used).
Several websites have written about using SDS and diluted acidic solutions for degrading Plasmid DNA in electroporation cuvettes. But I would like confirmation if such labmade protocols have worked.
Kindly suggest the percent of acid/sds along with any other components in the solution I would have to make.
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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.
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I am interested to pursue my study regarding antihistamines but I am not well-versed with molecular techniques. One of my aims is to analyze the long term effects of antihistamines on H1 receptors but I'm having a trouble on finding molecular techniques to use.
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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.
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I read several papers where researchers use several strains of the same species for analysis of recombination or selection pressure however there are few papers in which researchers use the multiple species belonging to one 'Genus', not the multiple strains that belong to one 'Species'. What difference is expected in the result and how it can be interpreted.
I want to know how my pathogenic organism 'Xyz abcde' is evolved (recombination and selection pressure). 5 different strains of 'Xyz abcde' is sequenced while 9 organisms in Genus 'Xyz' is sequenced.
Which dataset I should use to proceed and why? How it will make difference in the analysis?
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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
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I graduated with a bachelor’s degree in science in public health and nutrition. I considered pursuing a master's degree in medical sciences ( biochemistry and molecular biology track). What are my career options? And does it worth it?
Please, I need to hear from people from the work field. Just give me your opinion. 🙏🏼
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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.
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Suppose, I have a treatment group and a control group. I have measured the relative expression (delta delta ct) of two genes by real-time PCR analysis. Both the two genes are analysed from the same samples and the same reference gene was used. In this case, if one gene has higher relative expression than another gene, can I say that gene is highly expressed than the other gene. My question is if the relative mRNA expression from two genes can be compared? another question is, is it ok to produce a heatmap from relative expression data of different genes?
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Thanks a lot for your answer and suggestion professor Iman Hassan Ibrahim and Dr Abhijeet Singh
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I need to sequence an entire bacterial gene (6.1 kb). Is there a way to do it other than amplifying several smaller overlapping fragments for Sanger sequencing? Can I Sanger sequence the whole amplified gene using "walking primers", and if so, how is it techincally performed?
Is there any other method to sequence a single gene?
Thanks
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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
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Since the target region in the PCR is smaller than the template DNA strand to be copied.
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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
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1. How Long does it Take for Tamoxifen to activate Cre and knock out the gene of interest
2. What would the dosing regimen be for rats/mice?
I am designing an experiment where I will be knocking out 5-HT2A receptors in a cell type-specific manner before psilocybin administration to determine the necessity of 5-HT2A receptors in psilocybin's neuroplastic effects.
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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.
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Hi!
I have some DNA sample cut from streptavidin using excess biotin (30mM) and heat. will the residual biotin inhibit downstream PCR?
Thanks
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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.
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Hi!
I am about to use 95% formamide, 10mM EDTA buffer to elute biotinalyted DNA from streptavidin bead. So should I just simply add 1ml of 1M EDTA, 4ml of water and 95 ml of formamide to present 100ml of the elution buffer?
Thanks!
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Hi!
It is known that formamide and SDS can both disassociate streptavidin-biotin bond. Yet for most streptavidin beads, formamide are suggested to elute biotinylated DNA while SDS to elute biotinylated protein? What is the reason not using SDS to elute biotinylated DNA?
Thanks!
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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.
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Hi!
I am using a reverse transcriptase called "Maxima H minus" (Thermo#EP0753). Its protocol recommend using the Thermo Scientific™ RiboLock™ RNase Inhibitor (#EO0381). However, our lab are rich in stock of Takara recombinant Rnase inhibitor (2313A) and it takes too long to wait for the EO0381. What is the difference between the two inhibitor and can I just simply replace the Thermo Scientific™ RiboLock™ RNase Inhibitor with Takara recombinant Rnase inhibitor?
Thanks!