Protocol Design - Science topic

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

Get in touch for more info.

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.

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?

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

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.

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?

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.

A protocol for cell suspension in tomato (of any strain), priority to initiation from callus.

Appreciate all responds.

Thanks

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 :)

I'm looking for some standardized protocol for isolation of dna from cordyceps sinensis for further use in genome sequencing. Suggest if any ??

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.

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.

Thank you for any assistance you can offer

I am looking for a protocol to prepare high yield of EPS.

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?

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.

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

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

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

Can anyone provide change in code for ETT as a metric when we simulate RPL PROTOCOL in CONTIKI OS

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?

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!

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.

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!

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!

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.

I want to extract lipids from plant tissues for GCMS. Can anybody suggest the better protocol for it to get a high throughput?

Oxaloacetate (OAA), Phosphoenolpyruvate (PEP)

HPLC, LC-MS, Spectrophotometer

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.

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.

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

I am looking for a protocol to isolate unknown viroids from plant samples. Just wondering if there is any kit available for this

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

RPL CODE PLEASE

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?

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.

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?

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

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.

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.

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

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?

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.

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.

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

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?

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

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.

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?

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.

Aim to publish a medical simulation-based protocol. 

what is the best donor heart preservation method  for long duration and better function? ?

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.

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?

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

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!

For expression studies.

hello,,

Whats the difference between body sensor network and wireless sensor network in term of security?

thank you in advance

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?

I shall be grateful if any expert would share us the formula to estimate the study sample for a comparative study please.

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.

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?

What should be the concentration of RNase to remove RNA from DNA? And after removal of RNA how to denature RNase from DNA sample?

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.

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!

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?

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.  

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 

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.

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.

I want to pegylate HSA for my experiments.

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.

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.

PCR protocol.

Best simulator for MAC protocol design in underwater sensor networks.

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.

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?

.