Questions related to Molecular Biology
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?
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.
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?
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?
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.
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?
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?
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?
On the molecular biology level, what is the authoritative method to distinguish Lactobacillus casei and paracasei (or whatever they are called now) ?
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.
I looked up in the internet but couldnt find the antibiotic resistance. Someone knows? seems to be an oocyte expression vector...
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?
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?
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?
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?
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.
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.
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.
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.
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.
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.
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.
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.
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?
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!
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..
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.
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.
Thanks for all your help.
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!
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.
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...
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:
mTagBFP2 - Subach et al. 2011
(mCerulean3 - Markwardt et al. 2011)
mCherry2 - Shen et al. 2017
TagRFP675 - Piatkevich et al. 2013
FusionRed - Shemiakina et al. 2012
Would anybody be willing to share them?
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?
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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?
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....
"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.
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?
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?
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.
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
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?
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 ...
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.
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)?
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 .
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!
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?
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?
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?
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?
heating in formamide buffer is a common elution method to disassociate streptavidin. Yet is this disassociation irreversible when afterwards formamide is diluted and cooled?
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
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.
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.
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
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,
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.
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.
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?
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. 🙏🏼
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?
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?
Since the target region in the PCR is smaller than the template DNA strand to be copied.
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.
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?
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?
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?