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Hi guys, I hope you all are doing well
I just wanted to know wheter i can publish an experimental protocol design based only in previous literature (I have not performed it experimentally). If so, could you recommend some journals which I could submit my paper? It is a protocol for studing the intracellular and extracellular responce of human neutrophils against a yeast.
Thank you in advance
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It is difficult to publish a prposed protocol, framework or even a model in any journal without at least a pilot experiments. You should proof the applicability and efficiency of your concept with some results.
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Get in touch for more info.
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interested
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Though I am following a school on Youtube I am still wondering f there is anything that could be useful to have a basic understating of this disruptive protocols designing tool.
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Hello, if I understand what you mean, the following text may help you.
Zero knowledge proofs are proofs that fully convince that a statement is true without yielding any additional knowledge. So, after seeing a zero knowledge proof that m has a factor ending with 7, you’ll be no closer to knowing m’s factorization than you were before. Zero knowledge proofs were invented by Goldasser, Micali and Rackoff in 1982 and have since been used in great many settings.
The notion of proof is central to so many fields.In mathematics, we want to prove that a certain assertion is correct.In other sciences, we often want to accumulate a preponderance of evidence (or statistical significance) to reject certain hypothesis.In criminal law the prosecution famously needs to prove its case “beyond a reasonable doubt”.Cryptography turns out to give some new twists on this ancient notion.
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For example, Roche has discontinued its Universal Probe Library Products and the Assay Design Centre (https://lifescience.roche.com/global_en/brands/universal-probe-library.html#assay-design-center).
Changing the manufacturer can potentially change the previous results done using those products. Researchers whose previous researches were conducted based on those products will have to validate all those results again using new products from a different manufacturer to continue their researches. This can waste lots of fundings.
What are your opinions regarding this problem?
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Cameron Hyde , thank you so much for sharing all this info.
We want to continue using the UPL system in our lab and I'm just wondering about the reference that they "contain" LNA. Any idea if it is all of the 8 nucleotides? I'm thinking ordering a DNA only probe will have an impact on hybridisation efficiency/specificity.
Thank you all for this great thread.
Product discontinuation as in this case is a serious problem and IMHO the company should just share the product info freely so the community can keep working as before.
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Dear All
I am looking for a review which is interested in publishing protocol design. However, my study is not a random clinical study but a cohort study.
thanks in advanced for the help
Best regards
Karim
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Plos One, BMJ and trial
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Has anyone got any experience with diluting Cell Titer Glo before use, it's quite expensive and I'm thinking of diluting it down before adding to cells.
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Hello Joe Jones
Usually, Cell Titre Glo reagent is added in a volume which is equal to the volume of the cell culture medium in the well. You can dilute the reagent. But you need to be extra careful with the dilution as too dilute the Cell Titre Glo reagent, could affect the luminescent signal and subsequently the performance of the assay.
Best.
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Based on www.toda.network , the TODA protocol enables ledger-based blockchain to scale to billions of users with the near-zero cost for each transaction!
Now, who knows this ability on Industrial IoT networks in order to provide affordable solutions?
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Dear Elnaz Ashena,
Look over the following sources:
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I'ld like to know what are the effective ways of writing lab protocol ?
The one i prepared is attached in the Pic, I'ld like to know what all the other things that i can include to make it more effective.
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I am not sure of your objective. So I am sharing on general guidance. I think you have missed to include standardization of equipment's which is a critical one in protocols. If you are collaborating with others check with their validation sheets to match yours. I have had issues with flow cytometry results with our collaborators that are very different from ours. Also if you are using standards, the manuf & expiry date, consistency of pdt, etc. A better protocol is one which has clarity of each step, has repeatability and reliability. To document commercial reagents in a standardized way, I use RRID portal.
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A protocol for cell suspension in tomato (of any strain), priority to initiation from callus.
Appreciate all responds.
Thanks
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Yuan-Yeu Yau
Yes.
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Dear Research Community,
I need to write full research protocol and I need help in suggesting the steps to follow and the template for it please,
your guide is much appreciated
Mohammed Abed, MD
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Driss Benattabou
Thank you so much
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Hello everyone.
I want to lyophilize my plasmid DNA samples for long-term storage(>5 years) but can't find a complete optimized protocol for this aim.
Does anyone have a protocol or related reference for that?
Any useful information would be appreciated :)
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You can store plasmid DNA frozen in solution for years and years so long as it is not subjected to repeated freeze-thaw cycles (not in a frost-free freezer). Storage at -20 is fine but -80 is also fine. You can also store dried plasmid for many years without worrying much, just store in the dark so you don't get light/UV damage. You don't need to do anything special beyond drying it through Etoh precipitation or lyophilization. I think the main issue is whether you are just storing a stock away and would intend to retransform and make new plasmid prep in the future (in which case some nicking is not really an issue) or whether you need to store the DNA so that it is ready for experiments some years hence, in which case you may need the extra precautions described above.
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I'm looking for some standardized protocol for isolation of dna from cordyceps sinensis for further use in genome sequencing. Suggest if any ??
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The Qiagen DNA extraction kit works great for fungi. Just make sure that you grind it really well to disrupt the cell walls.
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Dear researchers,
I hope you and everyone in your circle are doing well and healthy.
I’m going to work on some factors that affect on differentiation of cardiac progenitor cells to cardiomyocytes.
I’ve collected stem cells from mice and now I require the best and cheapest protocol for their differentiating to cardiomyocytes. As I am an amateur in this field of research, I hope you can suggest a useful protocol.
Thanks all.
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Dear Soudabeh,
In this article (attached), there are several protocols treating your topic. I hope it will help you.
Best wishes
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Hello all,
I am attempting to determine the concentration of B. diminuta bacteria in an inoculated broth after 24 hours at 30 C on a shaking incubator by performing a spot plate of diluted samples and back calculating the original concentration. When I perform this, I am consistently getting very high CFU/mL values of approx. 1x10^11 CFU/mL. Can someone look over my experiment and tell me if I am making an error somewhere or is this value correct?
1) Inoculate 500 mL of Saline Lactose broth with inoculating loop dipped in pre-prepared glycerol stock of B. diminuta. Incubate on shaking incubator for 24 hrs. at 110 rpm.
2) Make eight 10x serial dilutions from 10^-1 to 10^-8 using 5 mL in 45 mL peptone water (1 g/L).
3) Plate 20 uL of each dilution in duplicate on Tryptic Soy Agar plates, allow to dry for approx. 5 minutes, and place in static incubator upside down for 18-24 hours or until colonies are visible for count.
4) Count the average # of colonies per dilution and calculate the CFU/mL using the following calculation: CFU per ml = Average number of colonies for a dilution x 50 x dilution factor.
Example: 10^-8 dilution average colonies: 19, (19 x 50 x 10^-8) = 9.5 x 10^10 CFU/mL.
Thank you for any assistance you can offer
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As Michael said, 10E11 cfu/ml seems well beyond what one would expect.
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I am looking for a protocol to prepare high yield of EPS.
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it depends on your EPS. for example xhantan gum extract with alcohol
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I was trying to electroporate my endometrial organoids using the NEPA system following the Fujii et al, 2015 (https://www.nature.com/articles/nprot.2015.088) and but so far no organoids established after the electroporation or they died due to the selection pressure.
Does someone has experience with electroporation of organoids or with the NEPA system?
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Hi Magdalena,
Did you solve it? any suggestions? Thanks!
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I am working with a complex plant material and I want to test different enzymes on it and measure the changes in carbohydrates and proteins fraction compared to untreated material. I got no experience on such assays and I would like to address some questions:
a) Is the activity assay mandatory for such experiments? Or declared activity is sufficient?
b) Since the material is in a form of sludge, what pretreatment is needed? Do I just add X ml of enzyme (enzymes are in aqueous solutions)?
c) Do you recomend pH adjustment with acid/base or buffer solution?
Thank you in advance.
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i) Enzyme assays are the established methods used for testing enzymes on a substrate or substrates. They are used to characterize a an enzyme by measuring its various parameters like activity, temperature and pH stability, Km and Ki etc.
ii) The pretreatment is required in case the material is insoluble.
iii) The pH adjustment can be done with both acid as well as base in different samples. That is going to depend upon the pH Optima of the enzyme being tested.
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Hello Esteemed Researchers
I have a question and I was hoping the experts in the field could guide me. So I have never worked with Shoot Apical Meristem (SAM) and am really curious to learn more and more about it in wheat. However, I do not know how to identify its location in a grown wheat plant. I tried searching articles which researched on SAM of rice and other monocots but the method of sampling or its location has not been stated.
I would really appreciate it if someone could advise me on this as well as explain to me thoroughly on how to identify the SAM region and what would be the best procedures to sample it.
Thank you so much in advance. Any form of guidance will be fully appreciated.
With gratitude
Dee
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Dear, Dee!
Typically, apical meristems are located at the top of the shoots (main and lateral). Such an arrangement of meristems is determined already in the initial phases of ontogenesis.
Perhaps, the following articles will help you by SAM in wheat:
Na-Sheng Lin and W. G. Langenberg. Distribution of Barley Stripe Mosaic Virus Protein in Infected Wheat Root and Shoot Tips
Ahmad, A., Zhong, H., Wang, W. et al. Shoot apical meristem: In vitro regeneration and morphogenesis in wheat (Triticum aestivum L.)
Wang PJ., Charles A. (1991) Micropropagation Through Meristem Culture. In: Bajaj Y.P.S. (eds) High-Tech and Micropropagation I. Biotechnology in Agriculture and Forestry, vol 17. Springer, Berlin, Heidelberg
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I am dealing with about 25 Alternaria brassicae single spore pure isolates since 2005. Presently trying to transfer them again on Brassica juncea and will isolate again to check their virulence. Its very difficult to bring back this pathogen on host even after maintaining 25oC and >80% RH with leaf wetness. Hope anyone will able to suggest perfect protocol for its pathogenicity test.
Thanks and regards
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Hi everybody,
does anybody have reliable protocol for measuring the protease activity? We would like to establish the protease activity from laboratory compost experiment (made from sawdust) and currently used protocol is not working.
The input material is wet sawdust and we probably use buffer which is not working properly with this material (PBS buffer pH 8 with Triton X-100). Then azoalbumin is added, mixture incubated at 37 °C, the reaction is stopped by 10% trichloric acid, centrifugated, add 1M NaOH and absorbance measured 440 nm. The measured absorbance is from 0-0,004, there is a possibility there is a low level of proteases, but we would like to proove the occurence/absence in our sample with different protocol.
Thank you.
Susan
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Hi Zuzana,
Please follow the link below that might give you some help regarding measurement of protease activity..
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Can anyone provide change in code for ETT as a metric when we simulate RPL PROTOCOL in CONTIKI OS
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Nice Dear C K Gomathy
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Hello, dear scientists
Can I seed cells with treating doses of the drug, and after that, add mtt reagent without removing media?
If I don't remove drug solution before adding mtt reagent, is it possible that drug solution interferes with mtt reagent?
Which one is better attaching with collagen or treating cells in suspension form at the first step?
Can collagen cause problems in the test?
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Greetings,
Although simple and easily reproducible, MTT assay technique have its limitation with regards to interference from treatment component. From my experience, my desired compound in the absence of cells did reduce MTT solution to formazan. Therefore:
1) It would be better to do optimization of MTT assay with your treatment component or;
2) You could substitute MTT with other reagents. If you are looking for cheaper alternatives, perhaps you can consider trypan blue exclusion method or Alamar blue assay for suspension cells.
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I have Heparanase III and Chondroitinase ABC which I would like to use to cleave the heparan sulfate and chondroitin sulfate chains off their core proteins before I run the samples on a western blot so I can visualise the core protein bands clearly (without the GAG-associated smear).
I have seen this done in many papers but there doesn't seem to be a specific protocol for when/how much/in what conditions/for how long the samples are treated with these enzymes. Is it best to treat the cell pellet before or after lysing them? Is there anything in a typical lysis buffer I should avoid? Or maybe it is better to treat the membrane itself after the proteins have run? How long/what concentration/what conditions should I use and how might I go about optimising that?
So many questions! Any protocols, papers with info or advice would be greatly appreciated!
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Kae-Jiun Chang
thank you so much for your detailed answer, it is very helpful! I will give that a try.
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Does Zigbee contain a field like NAV(Network Allocation Vector) which is present in Wi-Fi? The NAV field acts as a counter to tell the neighbouring nodes about the duration of the transmission.
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To answer this question one has to compare the different fields in the frames of the the two communication systems.
Best wishes
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Paraffin embedded staining is easy, but someone 'mentioned' that they thought it was harder in frozen cryostat sections. I would like to collect for frozen to expand the later uses of my tissue. Could anyone please provide a protocol or guidance on CC3 in frozen (15uM cryostat cut) section. Thanks!
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Dear Boobi,
Please, could you send me the protocol in case you did it.
Thanks in advance,
Sergio
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Hi all!
I am preparing to extract RNA from critical samples using Trizol and I also want to the proteins (and also DNA if possible) for later western blots.
Does anyone have special tips or tricks to this that are not written in the protocol?
I am extracting from a relatively small sample (mouse hippocampus).
Thanks in advance!
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I am a member of undergraduate research anticipating to investigate the protein expression of fungal biofilm after teatment of shockwaves and amphotericin B. After growing biofilm and containing its extracellular matrix in Tris-HCl buffer, a series of treatments, vortexing, ultrasonication, treatment with cation/anion exchange resins, centrifuging, and filtering with millipore membrane will result in supernatant that contains proteins, triglycerides, and carbohydrates. Unfortunately, we are unsure how to isolate proteins from the supernatant containing other organic compounds. Any feedback on how we can successfully isolate pure ECM proteins from the supernatant will be greatly appreciated.
In addition, if there are other successful protocols for extracting and isolating proteins from fungal biofilm ECM, it will also be very much appreciated.
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Hong-Guang Zha, Bubacarr G Kaira , Adron Ung , Achim Recktenwald , thank you all so much your insight and feedback to our question is very valuable and definitely adds more factors for us to consider for the course of our experiment. Again, many thanks!
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I want to extract lipids from plant tissues for GCMS. Can anybody suggest the better protocol for it to get a high throughput?
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Can anybody suggest me a better protocol for lipid extraction from bacteria sample (gram negative bacteria ) ?
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Oxaloacetate (OAA), Phosphoenolpyruvate (PEP)
HPLC, LC-MS, Spectrophotometer
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Thanks a lot for your valuable comment Sir Isam Eldin Hussein Elgailani
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I'm following Beaumont et al (2013) protocol of extracting collagen data. Has anyone faced a problem while demineralising archaeological dentin? Within one day, the dentin of one individual has almost completely dissolved into the solution! What should I do?I do not want to waste other samples.
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I am sad to say it probably means there is no collagen in your sample.
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I am suspending a pre-labeled lyophilized Amyloid-beta[1-42] in DPBS -Ca/-Mg. I have this now in 37degree incubator to aggregate.
The protocol I am following has further pelleting and rinse steps following the 48hr aggregation/incubation. The protocol is designed for 5ml tubes and says to pellet at 14,000rpm.
I do not know what rotor they used to do this, so I do not know the actual force. I hate it when people put rpm in a protocol and don't say what rotor.
Does anybody have an idea at what force I should do these spins? The reagent is pretty expensive and I am already late on this. I do not want to spin way too hard or way to easily.
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Sounds like a clinical centrifuge (Hettich, Eppendorf etc.). Their maximum rate typically is 14000 rpm, equivalent to about 20000-25000 g, depending on rotor geometry. Apparently, this centrifugation step is not too critical ;-)
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Hi all,
I have been told that you can take soil (it is currently frozen) and suspend it in water to then be able to take a swab (as bacteria can be hard to extract from soil), but I have not been able to find a protocol? I only have swabs and the Purelink extraction kit to use as doing skin DNA samples was my primary goal.
Help would be much appreciated.
Danica
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I tried it and it does not work as there are too many environmental influences and you need a soil kit to beat the DNA properly.
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I am looking for a protocol to isolate unknown viroids from plant samples. Just wondering if there is any kit available for this
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Hello everyone, I performed some lentiviral infections of adipose tissue mesenchymal stem cells to integrate an Dox inductible shRNA (pTRIPZ). however every time my cells are dying 2 or 3 days after the transduction step. My protocol is the next one:
First I seeded the MSCs on 6-well plate (50 000 ATMSCs/ well) and the next day I add my medium (MEMα, 10% FBS, 1% Pen/strep) containing my concentrate viral particules with 8µg/ml of polybrene and I spinoculated the plate during 1h at 270xg, 25°C. After 1h or overnight incubation , I replace the medium with fresh non infected medium. I usually got a good infection rate (GFP+ and puromycin selection) but unfortunately the cells stop proliferating and are dying after 2-3 days or 1 week. even If I pass them at 70-80% of confluence. In parallel, I also performed this protocol on ATMSCs but without viral particules as a negative control and these cells are proliferating and still alive so I am almost sure that the viral particules are toxic for the cells.
Is anyone meet a similar problem and solve it?
Thank you
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Hi Billy,
When you transduce with lentiviral particles, it is good practice to make a dilution series of your vector stock and transduce your cells with this series. Your titer might be to high for the cells and induce toxicity or to low making that not enough cells are hit by the vector (and the antibiotic selection marker is not enough expressed) . Also be sure that your cells do have the right confluency for transduction (this will depend of your cell type, but in general most adherent cells like some degree of confluency).
Another option is that the knockdown itself is toxic for your cells, but if I understand it correctly, you will have to turn it on. So probably, this is not the case.
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RPL CODE PLEASE
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check lines 84 to 94 of the code
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I am looking for a standard protocol of how to design a good primer for RT-PCR.
Could anyone recommend me the standard protocol of the design of (microRNA or gene) primers for RT-PCR as a reference?
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If you are looking for miRNA primer, then prefer pre-made primer from company. miRNA primer are universal primer sequence, which company synthesize with little chemical modification to for stability and to enhance specificity.
Rest gene primer can be synthesized using primerblast..
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I want to couple two proteins modified with NHS. I'm not able to find the protocol to couple these two proteins. Can anyone share the protocol with me. It will be really helpful for me.
Thanks in advance.
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Do you mean coupling one protein to another protein molecule? I have coupled rhodamine-NHS to a antibody,
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Who has ever used the first round amplified pcr sample as a template to perform second round pcr with same set of primers then run on a gel?
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This is something you should do only as a last resort. It breaks a fundamental law of PCR quality assurance, the principle of one-way flow. If you have to do it, mix your reagents in the pre-PCR lab, then add the product to be amplified in the post-PCR lab. Whichever way you do it, the chances of contamination are high. The more manipulations you do, the greater the chance of contamination. The last time I had to decontaminate a PCR lab, it took three people six weeks and cost the company 40 000 dollars.
Another problem is that primer dimer may take over your reaction. Dimers amplify more efficiently than correct product, so if there is even a trace there at the beginning, it will take over during the second PCR round. The less efficient and specific your PCR is, the greater the primer dimer problem will be.
Redesigning your primers using Primer3 or Primer Express and/or improving the purity of your template is almost always a better option. Simply diluting it often solves the problem.
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My research is focused on the development of an electrochemical paper based biosensor that is capable of blood plasma separation and the following quantification of proteins released into the bloodstream when traumatic brain injury occurs. I am currently developing the first part of the device following the protocol designed by Noiphung et al (2013, https://www.ncbi.nlm.nih.gov/pubmed/23845479) which uses the wax printing method for creating hydrophilic channels where plasma is obtained from whole blood by means of capillary action. However, having done a literature review, Kersaudy et al. (2013, https://www.ncbi.nlm.nih.gov/pubmed/23824514) mentioned this: “While paper can be used conveniently to separate several microliters of blood such as finger-prick volumes, it rapidly clogs and is unsuitable for larger volumes, limiting its use to the analysis of highly concentrated analytes, such as glucose or abundant proteins". The problem lies in the fact that the proteins I want to detect have an average concentration in serum of 0.3pg/mL and with my paper microfluidic device I could obtain a maximum of 100 microliters of plasma in the detection zone. Since the electrochemical essay comprises an antigen-antibody binding, I am not sure whether I will be able to achieve a detection limit low enough to detect the average protein concentration of 0.3pg/mL. Hence, I would like to know if someone is currently working on a problem such as the one I am currently facing.
I appreciate in advance your help. All suggestion are welcome.
Sincerely,
Francisco Burgos
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We were facing the same issue (achieving the low limit of detectionto by lateral flow assay). As we couldn’t solve it, we changed the approach to another method as of now.
Based on my limited knowledg, to achieve low limit of detection, you have to find an approach which is robust in detection of your analyte with regards to its molecular size e.g direct vs indirect immunoassay, enzymatic vs fluorometric, etc. (I chose competetive fluoro-based immunoassay).
Besides, you can think of amplifying either the detected signal or the analyte (i.e. your protein). For the first approach, you can concentrate your analyte in a n much smaller region. For the second approach, you can use a PCR (e.g. can integrate paper-based PCR into your current kit) to amplify the analye for easier detection.
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I have more than 7 experiences in statistical and epidemiological research which includes planning, management and monitoring of projects, developing research protocol, design of data structure, data management and analysis, quality assurance, graphical presentation of data and report writing. I am using statistical and data management software i.e. Stata, SPSS 17.0, Epi-info, Epi-Map, R3.2.1, MS Office, Excel.
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You are simply telling us your CV definetely but not a question.Thanks thou.
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I am following the vignette's protocol for the design II in AdehabitatHS using my data. But when I try to rasterize the polygons (14):
>pcc<-mcp(locs[,"Name"],unout="km2")
>pcc#it is a Spatial Polygons Data Frame showing the 14 polyogns
>image(maps)
>plot(pcc, col=rainbow(14),add=TRUE)
>hr<-do.call("data.frame",lapply(1:nrow(pcc),function(i){over(maps,geometry(pcc[i,]))}))
>hr[is.na(hr)]<-0
>names(hr)<-slot(pcc,"data")[,1]
>coordinates(hr)<-coordinates(maps)
>gridded(hr)<-TRUE
I got the following Error:
suggested tolerance minimum: 4.36539e-08
Error in points2grid(points, tolerance, round) :
dimension 2 : coordinate intervals are not constant
I would appreciate any suggestion on how to solve this problem.
I cannot figure out if this is a problem with my rasters (4 images) or with the Polygons, although I strongly believe this last ones are the issue.
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Thank you very much for your answer. the sp:: trasnform() worked well,
Thanks again
SGS
:-)
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hello researchers
i am working on an aptasensor based gold nano-particles , i need protocol to do immobilization of the aptamer on the gnps .. ..
any help please? and if there are guidelines i have to follow to insure good systematic experiment please let me know ?
thank you all
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thank you Jules
the gold nano particles are immobilized on the electrode surface
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We know that UC protocol is a gold standard for exosomes isolation. But there are many diffrent protocols used for it. Which steps do you recommend for exosomes isolation from plasma, saliva and urine. Is the optiprep gradient needed for cleaning the isolated vesiecles?
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Thanks for your answers 🙂
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I'm currently trying to evaluate B10 cells on spleen samples, but I was not capable of detecting IL-10 by flow cytometry at all. After isolating splenocytes I performed two protocols:
A) 6 hours stimulation on RPMI-1640 GlutaMax (Gibco)+ Ionomycin 500 ng/mL + PMA 50 ng/mL.
B) Overnight stimulation on RPMI-1640 GlutaMax (Gibco) + Ionomycin 500 ng/mL + PMA 50 ng/mL.
Brefeldin A (10 ug/mL) was added at the final 5 hours of incubation time on both protocols.
I don't know if this is relevant, but I'm stimulating cells on 6-well plate, 3x10^6 cells/3 mL.
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Your stimulation seems fine. Like suggested, monensin is said to work better for IL-10. I switched to using a mix of BFA and monensin when doing ICS, just so I am not missing out on CN signals. Cells seem to like it better when they are in round bottom tubes compared to flat bottom wells. So you could try plating them in 5 mL polypropylene round bottom tubes. Your cell concentration (10e6/mL) is fine.
Did you look at any other cytokines in your experiment (IFN-g maybe)? If you saw signals for IFN-g and none for IL-10, this means your stimulation/staining is fine and that you are not seeing IL-10 coz it is not there (your cells haven’t expressed any). What staining kit/protocol did you use? If you got signals for other cytokines with your reagents, it also confirms that the problem is not with the reagents you used.
While IFN-g and TNF are expressed at relatively high levels (TNF at 6-12 hours and IFN-g at 6-48 hours), IL-10 is not expressed as much and so chances are you will have trouble picking it up. In my experience, PMA + ionomycin stimulation induces a lot of inflammatory cytokines but not IL-10. If you are interested in detecting IL-10 for experimental reasons, try stimulating your splenocytes with LPS. Then try different time points (6 hours, 24 hours, 48 hours) and pick the one where you see the highest expression.
I found a paper which might be useful. They seem to be using PMA+ionomycin+LPS.
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I ran 48 samples from museum specimens on the MiSeq platform. Unfortunately, each sample has very low coverage (~0.0035x). One of the contributing factors appears to be that the average read size is 38 bp, which may be due to the large amount of sheering present in museum samples.
I was able to extract ~400 SNPs, but cannot produce any supported trees or STRUCTURE results. I know the low coverage is to blame, but I am not sure the minimum coverage that would be usable for an analysis. I typically see papers use coverages of >5x. Papers with lower coverage (the lowest I have seen is 0.5x typically mention that they would like to acquire further coverage).
However, to get that amount of coverage for one sample would require an entire lane of the HiSeq platform, which would provide 30x coverage than the MiSeq. If I went for 0.5x coverage, I could run 10 samples.
Is there a hard rule suggesting the minimum amount of coverage for a phylogenetic analysis? Is running these samples on a HiSeq worth the cost?
My species is Microtus ochrogaster, which has a reference genome (~2.3 billion bp). To prepare the libraries, the EcoR1 enzyme was used in a RAD-Seq protocol designed for degraded museum specimens. The amount of reads per sample varies with some alignment rates between 12 to 80%. Very few of the samples share SNP loci, with each loci missing >85% of the data.
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All samples, mostly skins, were extracted by a previous graduate student (Adams et al. 2017). The DNA for the most part is low-quality and low-quantity. Initially, I tried using Sanger sequencing on multiple loci, but the DNA was too poorly degraded. In the end, I could only get 500 bp of mitochondrial DNA from the samples and no nuclear DNA. In order to get nuclear sequences and additional mitochondrial seuqneces, I followed a RAD-Seq protocol designed for museum specimens by Tin et al. (2014) to build the libraries.
The goal of the project is to determine the validity of the current seven subspecies designation by sequencing samples from across the range of the species. Literature recommends that multiple loci from nuclear and mitochondrial DNA be used in a phlyogenetic analysis like this.
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Have a nice day every one
I’ve prepared slides for immunohistochemistery finished staining with DAP and Heamatoxyline and put the covers lip but unfortunately the staining was poor
I want re-stain the slides is there any protocols for this ???? I want to remove the cover slip (this is my problem) !!!!!!!
thanks
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Removing the coverslip is the hardest part. I soak slides in 60C xylene for 5-10 minutes to dissolve the mounting medium (I use prolong antifade). This loosens the grip of the coverslip. The tissue I use is paraffin embedded, but by the re-staining phase they are already de-waxed. Still I'm not sure if tissue embedded differently will be affected by xylene. (Xylene is a hydrocarbon, in this case used to solubulize hydrophobic mediums, like wax). If the coverslip is still tightly attached, I gently lift a corner with a pipette tip, and squirt some xylene underneath the slip to further loosen it. Then with fingers or tweezers pull it up. If it's not loose enough the tissue might get pulled up with the slip.
I've successfully re-stained IF slides with a second round of IF, or followed by periodic acid Schiff/Alcian blue stained. You can also follow FISH stains with PAS/AB, just not the other way around. I'm not sure how successful DAB/heamotoxyline re-staining would be, I've never tried it.
Hope that helps
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How easy is this to do for someone who has never done this before? We have plenty of experience culturing cells but how easy is it to recognize when it has worked? And does anyone have a simple, detailed protocol for us to follow?
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I want to see localization of mRNA in cell using DNA probe there r two alternative LNA probe and DNA probe,I want to know how we can design a DNA based fluroscent probe for mRNA detection ,if any one has used it kindly share protocol
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if using human cells try smart flare by millipore
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Hello,
I've been trying to induce expression of a recombinant protein (dimer, ~140kDa) in E.coli strain BL21pLYs in little culture volume (10-25mL) for a high throughput-like screening of mutants. I'm having trouble measuring protein activity, as sometimes the same protein variant express, and sometimes it doesn't. I can't find anomalies in the protocol that justify these changes in results. I know that expressing in small volumes it's not the best idea, but it would save a lot of time if i can make it work. Has anybody got any advice?
Protocol:
Grow starter culture (~1-3mL, 37°C) overnight from a glicerol aliquot of bacteria.
The next day, grow 100uL of starter in 10mL of fresh LB-medium in a 25mL erlenmeyer (or 250uL in 25mL of LB in a 50mL erlenmeyer) to OD(600)=0,6 at 37°C (3h.). Then, induce overnight with 1mM IPTG at temperature 20°C.
Following day: centrifuge cultures in falcon tubes. Discard supernatant. At these point pellet may be stored at -20°C for a couple of days. Then: Resuspend pellet in buffer (50mM Triss, 250NaCl, pH 7,5), concentrating 10X. Lyse cells in sonicator (3 rounds of 30" at 10Watt with 1min pauses between rounds. Tubes are kept in ice). Centrifuge at max speed and discard pellet.
Vector used: pET24A. Resistance factors: Kanamycin (25ug/mL) and Chloramphenicol (17ug/mL).
Thank you for your advice.
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A couple of ideas to explain the inconsistent results: inconsistent amount of aeration of the cultures, and inconsistent sonication efficiency.
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I am trying to quantify the number of Aspergillus species using 'absolute' quantification and I can't find many protocols for designing standards. Is it acceptable to use purified qPCR products as standards if I am doing a two-step RT-qPCR or do I need to generate RNA using in-vitro transcription to also account for the cDNA efficiency?
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You're going to be measuring DNA eventually anyway (because you convert your RNA to cDNA) so using a DNA standard is perfectly fine: making an RNA standard and then trying to convert it will (at best) give you "a DNA standard with extra error".
Many people suggest cloning your targets into plasmids and diluting those, but I don't see why this should be necessary: the vast majority of your amplification will be of PCR products, so why not begin with a PCR product?
So: run a PCR for your target, run the product on a gel, extract it, clean it, determine the concentration (and by comparison with the molecular weight) determine concentration in molecules per ul.
Then use this in a standard curve, from 10^7 down to 10^0 or so.
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Which method is more suitable for validation of circular RNAs; PCR or Northern blot? Can anyone suggest me the protocol of designing divergent primers for circular RNAs.
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cell free RNA once you isolate and convert it to cDNA then u can use normal PCR to validate only thing to take care is keep same primers will incrementing serial concentration of cDNA template and find optimal vol for getting positive band
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Aim to publish a medical simulation-based protocol. 
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Have a look at the Sage journal: "Quality Health Research"
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what is the best donor heart preservation method  for long duration and better function? ?
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Hi,
this article might be of interest for you.
Regards,
Nicolas
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I want to normalize the data I obtain from 96 well plate and I planned to normalize the data on the basis of cell number. Can you suggest me the methods which I can use to count my cells ? I will prefer  Non-fluorescence based methods to do that.
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Thanks Rahul for your answer but I think counting 96 wells manually will be too hard and it could also affct the accuracy.
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A study protocol is designed to detect the faults & physiological effects due to unidentified misplacement of DLT (inserted conventionally for one-lung ventilation) by using FOB. What may be the confounding variables in this study & how to deal with those?
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Rabaul,
I'm afraid you do not present enough information for a complete assessment of potential confounding variables. It seems to me that at some point the "unidentified" misplace net has to be identified to be entered into your study. Therefore, the entire premise escapes my understanding.
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i want to design IEEE 802.15.4 MAC protocols specific to GTS allocation which is also known as Contention Free period (CFP). suggestion about simulator which is suitable and i am new in that field how can i start recommended article or tutorials to start work. Thanks in Advance
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I would recommend NetSim (http://www.tetcos.com), since its got an extensive 802.15.4 library, and it would be very easy to modify CAP, CFP, GTS etc.
By the way NS2 is no longer under active development and is not accepted for publications is what the forums say.
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Hello,
I am using mRNAseq data from the TCGA database. They have used both RPKM and RSEM to normalize the read counts. I would like to use the RSEM normalized read counts for the study but if anyone has any reasons not to please include that in your response. Here is a link to a paper describing RSEM http://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-12-323
According to a previous post..... (How can I calculate z-score from rpkm or counts values? - ResearchGate. Available from: https://www.researchgate.net/post/How_can_I_calculate_z-score_from_rpkm_or_counts_values [accessed Jul 28, 2016].)....The process of calculating z-scores is as follows:
1. Convert the RSEM normalized read count values of each gene into log values.
2. Calculate the mean and standard deviation of log values for each gene across all samples in the data set.
3. Convert each log value into a z-score as follows: (gene X log value - mean of log values of all samples)/ standard deviation of log values of all samples.
The above protocol was designed for standardizing RPKM values, but will this approach work for the RSEM normalized counts as well?
Thank you for your help!
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Can follow this instruction Michele.Yes you can get the RESM normalization count.
calculating Z-scores is :
z = (expression in tumor sample) - (mean expression in normal sample)/ (standard deviation of expression in normal sample)
For RESM ,
RSEM data in TCGA studies are diploid samples.
z = (expression in tumor sample) - (mean expression in diploid samples) / (standard deviation of expression in diploid samples)
Given below the links. I hope you will do your work
Regards
Md. Julkar Nayeen Mahi
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For expression studies.
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hello,,
Whats the difference between body sensor network and wireless sensor network in term of security?
thank you in advance
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General sensor networks has a typical wireless concerns for massive small devices, and sensor network issues have been investigated for years (military and environmental civilian purposes). Body system has the sensitivities of safety (it may deal with human body and medical problems; e.g., over heating may be a big problem) more privacy is needed (association with individuals), and DoS attacks may be deadly (literally speaking), and so on. A good start for the latter subject and its component you can find in: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274043/
(It is a standard so it has some general material and some general issues, general but a good start).
Wireless security in general has numerous books and many many papers on.
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Also, I want to ask if I worked with agrobacterium I need to design a special plasmid for my fungus transformation or I can use the plasmid used with other species?
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I shall be grateful if any expert would share us the formula to estimate the study sample for a comparative study please.
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Dear Dr.Rabiul Alam.After reviewing the valuable answers of the researchs who answered your question and reviewing the article I cited for you, I am confident that the article  will answer your question.
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In generic secure computation protocols, garbling have to be followed with the oblivious transfer. Can anyone suggest me the best two-party OT protocol? Implementation details will also be appreciated.
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I don't know what you mean "best" but one of the most elegant and simplest I've seen is this: Tung Chou and Claudio Orlandi: "The Simplest Protocol for Oblivious Transfer"
The implementation is far too simple to discuss it.
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I plan on evaluating PBMC supernatants with the Biolgend Legendplex bead array kit (13 inflammation panel - IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-γ and TNF-α).
Do I need to have separate wells/plates for each time point collection?
Would the longer incubation periods (48 and 72hrs) require fresh medium added to the PBMCs to keep them going?
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Thank you Olga.
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What should be the concentration of RNase to remove RNA from DNA? And after removal of RNA how to denature RNase from DNA sample?
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i extracted DNA from soil samples and Now I need to treat purified DNA with RNAse. the DNA volume is 70 ul . what are your suggestion without  losing my DNA ?
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I want to know activity of glycerate kinase from my microbial culture. I am not able to find out suitable method to do it properly. Please suggest me better protocol for analysis of this enzyme.
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Hi there,
Glycerate kinase activity can be easily coupled to pyruvate kinase and lactate deshydrogenase. GK activity will result in the stoechiometric consumption of NADH measured at 340nm. For more info this paper should help :
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I have tissue parafin embedded sections and I would like to co-stain for Tunel (Roche kit) and F4/80 macrophage staining.(Flouresent).
I would appriciate if some one can give me a reliable protocol.
Thanks!
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since Tunel kit that i have is In sito detection kit i don know if i can combine it with another staining which need blocking!
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I need to measure the indoleamine 2,3-dioxygenase (IDO) activity in cultured adherent cells. In some protocols measure the enzymatic activity in after lysate the cells and in others in the supernatants. Which one is the correct way to do the assay? Also, I have read that some protocols after addition of trichloroacetic, the plates were incubated at 50°C for 30 min to hydrolyze the N-formyl-kynurenine to kynurenine, but others do not. Do you know the best protocol to measure indoleamine 2,3-dioxygenase (IDO) activity in cultured adherent cells?
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Maybe it is too late.. But, please check the attachments..
Regards,
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As the mostly WSNs are event-driven system, efficient event-detection protocol design is recently growing research directions. Any information will be given on this, will be matter of appreciation.  
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Thanks Dr. Shen for referring my survey paper. Rajeev, you may find the paper here in Research gate as well:
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When we mention that Study duration was 4 months  does that mean it was time taken to complete data collection or  it takes account the time since inception of the protocol or design of study to end of data collection
IF possible can anyone attach reference to it 
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The study duration is of data collection starting from commencement of data collection till end.
In this regard, the pre-trial preparation or pre-study preparation as well as statistical analysis and reporting are not considered.
For example, calibration of an instrument to measure some biochemical tests before the trial started.
The study duration was 2008-2011 during which we collected data, the statistical analysis and publication took time. In 2008, we started collecting data and till 2011 the data collection continued.
Before data collection, there was a period of few months during which we procured kits, contacted schools etc.
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To identify agarase-expressing bacteria within a mixture of Escherichi coli strains, samples of E. coli were serially diluted and spread over LB (Luria-Bertani) agar plates. After overnight growth, zones of hydrolysis surrounding individual colonies were stained using potassium iodide, at a final concentration of 1 mM.
Urgently, thanks.
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I do not have a written out protocol. But roughly: an overnight E. coli culture in LB will be around 1 x 10E9 cells/ml. Single colonies are accepted to be true single colonies if there are between 50 and 300 on a 9 cm petridish. So when plating 100 µl you need a dilution of about 10E6 To compensate for innacuracy in the initial estimate work around that e.g. 10E5 - 10E7. The attached scheme is for plating starting from soil samples but it illustrates a bit what I mean. 
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I want to start some in vitro exp. with bronchial tissue cell line, but I dont have som experience with this topic. Before I worked with fibroblast cell line, so I have some work skill with the culture and all work around. Please help me, the best with protocol. Thanks.
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Not my area of expertise. Sorry
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I want to pegylate HSA for my experiments.
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Thank you Robert!
I am currently following the protocol from the book you mentioned. :)
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I want to analyze different routing protocols for wsn. I have previous experience in dealing with ns2, but after setup ns2&mannasim, I found just leach and diffusion protocols. I ask if there are other simulators with source code for other routing protocols designed for wsn.
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I induced arthritis to my mice strain. I would like to get some protocol or know how I could isolate cells from the joints and be able to do a FACS analyse and not just an histochemistry.
Thank you so much for your help.
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Dear Natalia,
You need synovia infiltrated cells. Before FACS analysis, you should handle single cell suspension from synovia by mechanical or enzymatically degradation method. You can use Medimachine  device for mechanical degredation of tissues.
Good Luck
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PCR protocol.
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Depends on the species. If you are interested in P. falciparum, you may want to use reduced annealing and extension temperatures because of the AT rich genome.   We generally anneal at 47 oC and extend at 2-4 oC below the melting temperature of the primers. 
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Best simulator for MAC protocol design in underwater sensor networks.
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You can use following simulators based on your specifications for conducting experiments.
NS-2, QualNet Aqua-Sim,  and UWSim but I can go with  either UWSim or Aqua-Sim. If you are beginner  then you need to go with Usim that requires a PC running GNU/Linux and with a decent graphics card (i.e. NVidia or ATI). If you possess intermediate knowledge of handling the complicated scenarios in Underwater then you should go with Aqua-Sim that is easy to use with enough features supported by NS2. Aqua-Sim  simulates the attenuation of underwater acoustic channels and the collision behaviors in long delay acoustic networks. Moreover, Aqua-Sim
supports three-dimensional network deployment and provides a healthy set of basic and advanced protocols.In NS-2, Aqua-Sim is in parallel with the CMU wireless simulation package.
Aqua-Sim follows the object-oriented design style of NS-2, and all network entities are implemented as classes in C++. Currently, Aqua-Sim is organized into four folders,uw common, uw mac, uw routing and uw tcl. The codes simulating underwater sensor nodes and traffic are grouped in folder uw common; the codes simulating acoustic channels and MAC protocols are organized in the folder of uw mac. The folder uw routing contains all routing protocols. The folder uw tcl includes all Otcl script examples to validate Aqua-Sim. Users can use the examples as templates to conduct  new experiments.All classes related to MAC layer are included in the folder of uw mac. There are four MAC protocols: Broadcast MAC,
Aloha, Tu-MAC and R-MAC are implemented in Aqua-Sim. Aqua-Sim can be used over Unix and Ubuntu operating systems.
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Whenever I am using my present protocol, the DAPI staining is diffused throughout the entire tissue section. This leads me to believe that my antibody stain results are unreliable and I need to tweak my workup protocol.
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Yes prolong Gold is so quick and easy to use. Try dapi stain on section before immunostain. Could be something in your method that is degrading the DNA
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I'm currently trying to adapt the techniques from one paper that outlines LC-MS analysis to the machine that we currently have.
In the paper that I'm using as a template the group uses a 2.1 x 50mm C18 Column packed with 5 um particles, a flow rate of 1mL/min, and a sample injection of 10-50 ul.
However the LC-MS that our lab has formation runs of 0.15 x 150 mm C18 column with a flow rate of 1 ul/ min,
I've looked heavily through the literature but I haven't been able to find anything that discusses how to adapt protocols for different column considerations. How would these things scale for our lab's device and what are the major considerations that I need to keep in mind when planning the experiment?
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In addition to Farooq's points: column capacity (scales with diameter2, 1/14 column diameter will roughly translate to 1/200 sample load), injection volume (not good to inject 50 ul at a flowrate of 1 ul/min), dead volume/dead time of tubing (calculate but also determine experimentally by looking at injection peaks), detector sensitivity and linear range (determine via dilution series neat and in matrix), reequilibration time (scales with column volume).
At the lower flow rates the MS response should also increase, since the 1 ul/min flow rate is much closer to the sweet spot of most ion sources (5-10 ul/min).
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.
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Hi,
Since MCF-7 cells require the estrogen hormone for their growth, Beta-Estradiol pellets are used for the artificial release of this hormone.
You can follow the following protocol for preparation of your xenograft mo