Questions related to Molecular Biology
I would like to buildup a small active research group including a comprhensive subspecialities in Clinical Biochemistry, Molecular Biology, Internal medicine, statisticians to be shared in writing research articles, review, chapters and books. Who see him a suitable he can comment here with his email or whatsapp no to communicate later.
Is there anyone who has done TaqMan assays using average regular use PCR mastermix (not the TaqMan assay specific mastermixe) using cDNA as template for the qPCR test? I wanted to know the ins and outs of the procedure and the optimization you did to get accurate results.
Thanks in advance.
Hello every one,
I'm working on the in vitro expression regulation of a viral gene.
I'm looking at the splicing efficiency of several versions of the gene (comming from different genotypes). To do so I cloned the various versions of the gene in a pcDNA3.1 vector.
When expressed in eukaryotic cells, all vector expression but one give me the expected splicing pattern.
The odd one use a new splice site really close to the CDS start never reported in the litterature and absent in all other version of the gene.
The gene in the pcDNA3.1 is under the strong pCMV promoter which also add a 150ish base pair 5'UTR to the mRNA. Whereas, in vivo viral mRNA have a very short 5'UTR or none at all.
I was wondering if the 5'UTR addition and/or the strong expression could suffice to force this artefactual splicing ?
Thank you for your time !
I am currently optimizing a RPA-based protocol. As everyone knows, this nucleic acid amplification technique is based on isothermal amplification (around 37-42˚C) in combination with recombinases and single-stranded DNA binding (SSB) proteins.
I have designed several candidate primers to optimize my RPA. All of them were searched in literature and designed considering the criteria to be used in an RPA reaction.
These same primers were verified by conventional PCR (At different Tm: 55-65ºC), obtaining a successful result.
The problem started when I used the RPA master mix (lyo version from Twistdx) and performed the RPA reaction with the same primers and samples used in the conventional PCR. ALL THE RESULTS BECAME NEGATIVE?!
Primer concentrations used in RPA reaction was 400nm (recommended by twistdx) and the reaction time was 30 minutes at 39 °C (conditions recommended for the set of primers tested). Visualization of the amplification was done on agarose gel (1.5%) and doing a posterior melting curve assay. In all cases, no amplification was detected.
Does any one have a clue on what is happening???
I don´t know which other variables I can change to obtain good results
I am very curious about the ecology of Chroococcidiopsis thermalis PCC 7203. Does someone have an information about the chemistry of a spring and its temperature where Chroococcidiopsis thermalis PCC 7203 had been isolated from? Who did isolate this culture? Thanks. IB
I'm struggling to publish my work because I cannot find a good journal with less publication fee as most of the good journals are charging more than $2000. My work is based on colorimetric LAMP assay with a novel fluorescent dye.
Hello to all my fellow Biologists.
I have resorted to posting this question as my last desperate attempt to find a place for myself in the world of biotechnology.
I am a first class student with relatively good grades (GPA 3.77/4.00) in BSc. of Biotechnology (Hons), excellent extra-curriculars and competitions under my belt, 6 months of work as a Field/Research Assistant and 4 years of previous work experience in events management. I have experience in the microbiology, molecular biology, antivirals, nutraceuticals and cell culture disciplines. I have also taken a great liking to scientific communication and create visual content to make biology simpler for the Layman, both bother and in my own time.
Despite all this, I haven't had a single postgraduate application succeed for the last 2 years. And though I do understand there is high competition for available spots, I also wonder what I may be lacking despite some telling me I have an "impressive CV" and can do a direct PhD.
It is unfortunate however that my family is not doing financially well, therefore I can only afford opportunities with a scholarship or that are work/salary-based. Perhaps this narrows available opportunities but regardless, studentship scholarships have very evidently not opted for me, simply because "there were too many applicants this time around". Perhaps lacking funds is not enough of a criteria? (Hint of sarcasm).
Additionally, I was born and raised in the UAE (I do not get citizenship), therefore I am also looking for a potential country to eventually settle down in while doing the work I love.
I would greatly appreciate if anyone would know of opportunities I may be able to apply for like fully funded PhDs, or skilled/summer programs and workshops/internships, or even Research or Lab assistant positions you or someone you know may be looking for, because unfortunately, I'm 2 rejections away from being completely out of options.
I would greatly appreciate any input you may have or can share with me! I have also added my CV for your reference.
I don't want my impression of the field I love to be tainted with nothing but rejections, and to settle for a job outside our field simply because I had no other choice.
I look forward to hearing from you all.
Hi everyone, I've tried to transfer initially using a 20% methanol transfer buffer using 250mA max current for 70 mins, all I got was transfer of mid-high range proteins, I suppose all proteins below 20 kDa escaped the membrane from the other side. Anyone has any experience with blotting 5kDa +- peptides and can share tips?
A patient with desminopathy (mutation Thr341Pro DES in a heterozygous state) with the progression of the disease has a decrease in taste and smell, immunosuppression, and an increase in IgA in the blood.
Oddly enough, but all this is characteristic of infections, including viral ones. For example, it is known that if the hepatitis C virus is not treated, then death will occur in 20 years.
In the identified case of late onset desminopathy, muscle weakness manifests itself at the age of 30, and death occurs 20 years after the onset of the disease.
Could the desmin mutation in myofibrillar myopathy be caused by an infection?
Perhaps the infection contributes to the progression of desminopathy?
I know many websites have simple tools like transcription and translation available, but are there any analysis tools that researchers need that either do not exist or are not publicly available? It could be anything from algorithms to visuals. Thanks!
I’m trying to reproduce the library transformation efficiencies seen in this 2010 paper:
in which they claim 1.5 x 10^8 cf/ug DNA. My intact circular vector is the same size as their pcr product + linearized vector, but I’m getting 10^5 ish transformants/ug, similar to chemical transformation with the zymo kit.
Has anyone successfully done this? so far we’ve tried different electroporators, voltages, recovery media, etc but nothing seems to be working.
Any other ideas or troubleshooting would be much appreciated, thanks in advance
I would like to add BG-azide to cells for SNAP tag pulldown however I don't know whether it is going to be cell permeable. My instinct is to day yes it will be but neither me nor the supplier know whether that is true. I thought I would ask if anyone has tried something similar, even though that is unlikely... I have attached the structures in case that is informative
In a patient with desminopathy (mutation Thr341Pro DES in the heterozygous state) with the progression of the disease, we note signs and symptoms that are also characteristic of botulism: bradycardia, arrhythmia, AV blockade, a significant decrease in the average duration of motor unit potentials according to electroneuromyography, paresis and paralysis of the striated muscles, decreased sweating, paresis of the gastrointestinal tract, dry eyes, dry mouth, symmetry of neurological symptoms, hoarseness, impaired visual acuity, doubling of objects occurs, progressive muscle weakness. These signs and symptoms are characteristic of botulism, only when a case of desminopathy is detected, they proceed slowly.
I have protein with concentration of 0.6 mg/mL
The total volume of my protein is 4 mL.
Protein size is ~18 kDa
How can I convert my total protein concentration to micro molar?
I'm optimising a new RNA extraction protocol from yeast. The RNA extraction with formamide works great (the yield of RNA mass/YDM is even better than our established protocol) but the final volume of formamide in which the cells are disrupted makes the sample very diluted. I was thinking of ethanol or LiCl precipitation, maybe using SpeedVac (although formamide boiling point at normal conditions is 210°C so I'm not sure how possible this is; for two-phase extraction the only common solvent it doesn't mix with is diethyl ether which does not dissolve nucleic acids well). As the formamide extraction protocols don't seem popular, the published sources are very limited. Does anyone have experience concentrating RNA in formamide?
I have a tube of antibodies marked by the manufacturer that can stain frozen sections and paraffin sections for immunofluorescence, but the manufacturer did not indicate whether it can stain immunofluorescence of cells. I wonder if such antibodies can also be used to stain cells for immunofluorescence. Has anyone encountered a similar problem, looking for your answer.
I used to activate my PVDF with methanol for 5 minutes, then I did transfer as usual. But these days twice I got the same results, which is there is a block white marks in between after I finished my fast-green staining. I do believe those are not bubbles formation.
Some student suggest me to wash the PVDF first with water then continue transfer and so on.
I confuse, what's wrong with my technical transfer. I have never facing this situation before.
Hi I am looking for any alternative protocols to purify TMV virions, other than the commonly used PEG precipitation.
Two main problems that I have with PEG protocol is that,
1\ any macromolecule will co-precipitate as well
2\ so that PEG cannot purify full length (300nm) TMV virions from other shorter rods (probably breakage), and other small discs. (thanks to people responded to my earlier question)
So I am wondering if there is any purification protocol or method that can get mostly the full length rods and rid breakage ones and discs?
Thanks a lot in advance
Sometimes it may be difficult or expensive to carry experiments by yourself. If commercial companies can do that at a satisfactory cost, our ideas can be translated into experimental results faster and with better expertise. I was thinking if that can be done.
Sometimes we may try to do some molecular, immunological or mouse experiments if the experiments do not require specific patient samples and starting data can be emailed in soft copies.
If anyone have idea, please help inform me.
we recently switched most of our -80°C freezers to the absolutely scorching -70°C in my lab.
We did so because apparently the -80°C guideline originated from a technical limit imposed by the cooling fluid used back in the day. Thus it has nothing to do with a potential increase of our beloved samples stability over time.
It has no impact on sample life BUT the energy spent for going lower and lower in temperature increase exponentially. From what i can remember the increase in energy cost could be as high as 30%.
Here are some resources on the subject.
What do you think of that ?
I am finishing a Ph.D in biomedical sciences, I have a master in molecular biology and I know self-taught programming in Python and C. Do you think I could apply for a computational biology post-doc?
What could I do to be competitive? Would a Github with example of my codes and programming certificates from Coursera enough?
I was hoping that some of you might be able to help me set up an experiment in which I want to measure telomere length of MNCs (of a certain patient population) by Flow-FISH. As I'm completely new to this I'm running into a whole bunch of issues. If you have thoughts on any of them, feel free to comment.
First off, what probes are best to use? Traditionally people use PNA probes, but Exiqon also seems to offer LNA probes these days, are those any good? Others say Bridged Nucleic Acids (BNAs) are the new hotness. If sticking to PNA, who has experience for a good supplier for the EU?
I just want a general (though accurate) estimate for telomere length per patient. Should I get TelG or TelG probes, or both and mix them? Also, I want to use a green fluorophore, AF488 is a lot more expensive than FITC, are signals so weak that it merits the extra $$?
Assuming it's best to include a DNA dye to correct for pleudity, I was thinking of using LDS751, but considering the plethora of new dyes out there I'm open for alternatives. Will 7-AAD work for cell cycle analysis, or one the new patent dyes (RedDot2, DyeCycle Ruby)? It needs to be excited by the 488 laser and emit in the red spectrum as not to give too much overlap with my FITC signal. Also as little as possible excitation with the HeNe laser would be nice, as I need that for other stuff.
I want to use a RALA peptide vector, and I have found that some studies centrifuge the plasmid/RALA complexes and resuspend the pellet before use, while others seem not to do so. It seems like the studies that don't centrifuge end up with smaller particle sizes than the studies that do centrifuge, but I haven't been able to find any definitive confirmation of this trend. Intuitively, it seems like spinning them at 10k RPM would mash them together to some extent, possibly causing aggregation/melding of the particles and lead to larger particle sizes after resuspension. The only advantage to centrifuging and resuspending I can see is that it would eliminate any toxic effects of free floating/non-encapsulated plasmid, but this wouldn't even really be a concern in vitro, right? Does anybody know of a study that has investigated this? Thanks.
Good time, dear, I need a journal article clarivate about cancer, immunology, and molecular biology and is easy to accept. PhD student and final year thanks and best regards
I am using DNeasy PowerBiofilm Kit to isolate DNA from skin swabs. Now I am considering if I should use the provided collection tubes to final storage of extracted DNA. DNA can bind to plastic walls of the tube, so should I rather use low-DNA binding tubes from Eppendorf? On the other hand, the kit has been made for the extraction of DNA from various types of source samples, so it should be appropriate for DNA storage, despite nowhere is wrote that the tubes are DNA-low binding?
Hello, I am working with cell culture and I need some advice. I usually use trypsin to detach the cells from the flask, but I wonder if I can stop the trypsin reaction with serum-free medium instead of serum-containing medium. Is this possible or will it affect the cell viability and growth? How do you subculture your cells with trypsin? Thank you for your help.
In some cases the -10 of the promoter is known, while in others it is not.
In that case how do we predict the transcription start site (TSS)?
I have tried using the popular tools available. They give inconsistent results compared to some of the characterized promoters and transcription start site.
The bacteria I am using is Lactococcus lactis NZ9000.
Thank you in advance.
We are working on Biofilm and Quorum sensing genes of E.coli, we suggest some of reasons maybe:- Contamination, Annealing Temperature, Melting Temperature, DNA Extraction mistakes in the Techniques, we identified them by biochemical tests to be sure the isolates are E.coli, but now we are collecting samples from the beginning and doing the procedure again
I am designing a plasmid with an SV40 promoter-driven antibiotic resistance. Does expression from an SV40 promoter require a TATA box upstream of the transcription start site? The original vector had a TATA box at -30, however this is lost in my cloning strategy. With my current plan, the transcription start site is just 8bp from the end of the SV40 promoter. Will this allow for expression, or is a TATA box needed?
I need to do a primary culture of Non parenquimatic liver cells from mice and althought, I have the protocol for the obtaining and isolation of these cells, I do not know which medium to use, what porcentage of FBS use and what and how much supplements use (like Glutamine, antibiotics, etc).
I would really appreciate the help!
Could you please list some common methods for identifying the binding pocket? I mean we have a compound and a protein of interest, so which domain of the protein will be binding with the compound? And my major is molecular biology. How can I verify the binding pocket with the methods in my field? Thank you very much!!
I did a Gibson Assembly and after the assembly I stored them on ice, but forgot to put them in -20 degrees. This was at the end of the day. The next morning I saw that the ice was melted and therefore they havent been on ice but water for the night and part of the morning. When I saw this, I immediately stored them in -20 degrees. Now my question is: are they still good to use?
"The result shows absence of intragenomic variation among 16S rDNA gene and presence of variable regions among the 16S rDNA sequences (intergenomic variation), noticing for example high variability around 800, 900, and 1000 bp and a large conserved region between 1150 and 1350 bp. This information allowed us to discard the restriction enzymes FnuII, AsuI, FokI, Eco57I that recognized some restriction sites contained within variable regions, since they are more susceptible of acquiring future nucleotidic variations and with this, the potential generation of different band patterns." 
I add that the article mentioned that these discarded enzymes were targeting conserved sites in the study species.
Mandakovic D, Glasner B, Maldonado J, Aravena P, González M, Cambiazo V, Pulgar R. Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP. Front Microbiol. 2016 May 9;7:643. doi: 10.3389/fmicb.2016.00643. PMID: 27242682; PMCID: PMC4860512.
Is my reading right that the article implies that there is such potential? If yes, what are the possible mechanisms?
More important, what's the time frame of this "future nucleotidic variation", is it an evolutionary time frame that could take thousands of years?
Edit: i think my question can be thought of as: How common are new 16s rRNA gene variants in bacterial species?
I obtained the EnzChek peptidase/protease assay kit. I carried out the assay according to the manufacturer's instruction using a microplate and read at excitation/emmission of 490/520 but i got OVERFLW readings even for the negative control. Anyone with prior experience?
I am running a qPCR assay. I chose gradient temperature option for each of my primer to get the best conditions the amplification happens (without heterodimers- NA in negative controls). However, I have seen that my housekeeping gene and one of my target gene have different annealing temperature. Can I run another qPCR set-up just for this gene by choosing gradient temperature option ? For instance; my gene in question in a row with 54C and housekeeping gene in a row with 60C. I think as far as the machine reads the signals at the same time, it won't pose a problem but I just want to make sure.
One example would be MBP and MBP-74, an interaction which can be disrupted by maltose when it binds MBP.
My concentration was 123.5 ng/uL
260/280 Ratio - 1.725
260/230 Ratio - 0.401
Can I synthesize cDNA from the above RNA?
Need suggestions please.
I have been using NEB Hifi Gibson assembly for a couple years now and I've been quite happy with it. I regularly make plasmid constructs with 4-8 fragments, and always >1/4 of the colonies are "perfect," while the remaining ones may have some SNPs at the joining sites, or be misassembled due to a repetitive region.
Some colleagues said that Golden Gate has even higher efficiency. That even with 8 fragments, it is normal for 50-100% of colonies to be perfect. Is that true? Is Golden Gate that perfect?
I’m currently working with those two vectors and need to check them via PCR, but I’ve been unable to find their sequences online so far. If anybody has them I would be really grateful if you can send them to me or direct me to an online source.
Thanks in advance!
I have two sequences from the predicted mRNA sequence (only exons, without intron) and gDNA sequence (with intron). Then, I align the sequences to confirm the position of exon in the DNA sequence. after that, I pick the primers from the exon region and check the specificity on Primer Blast. However, I also design primers only from predicted mRNA without considering the exon region on DNA sequence. Which is more appropriate to use in amplifying full-length genes in the DNA template?
I am currently working on Co-IP of NRIP and MyoD by overexpressing both proteins in HEK293T cell. The question is I cannot get a specific band of MyoD. For my input, I think the protein level is too low to be detected so I will increase the protein amount to run a western blot. However, several bands appear when detected using MyoD antibody after co-IP. The band most likely MyoD is pointed with a blue arrow. Some of my colleagues say it may be endogenous MyoD or protein with similar conformation. Does anyone face a similar situation when working with this protein? or any reference paper suggested for studying this protein. Thank you.
i am new to enzymology and want to study the discipline through history based books (i,e, how was the Kreb's cycle discovered, methods used then, etc). what are your suggestions to achieve this?
better to be from wiley or springer.
Can alpha glucosidase and alpha amylase work at room temperature and at 37*C? If so why? The specifications for these enzymes are that they work at 37*C. But number of papers have modified their protocols 25*C. How can it work at both room temperature and at 37*C?
If protein expression in blood monocytes was low (detected by western blot) and protein level in serum (detected by ELISA) was high? what dose it mean?
Note: the protein should not leave the nucleus because it’s a DNA-binding protein.
Please see the attached image for the reference. I have been seeing these cells in the Arabidopsis thaliana seed (post germination) whenever I go for microscopy. The cells are attached to seed coat and they are alive for more than a week.
I did a google search using image and got no relevant results.
I am very curious what are these cells and I will be thankful if could suggest me what is the term used for these cells or any other relevant information.
I am looking for a method to completely remove proteins from plasma without using heat or adding salt ions. I have considered using activated carbon, but I am unsure if this is feasible. Are there any other effective methods for achieving this goal?
What bioinformatics tools are available to help analyze and interpret large-scale molecular data generated from crop research?
Please help me with the SDS-PAGE issue. I cannot see the protein of interest but the dirty dots all over the blots. And the dots are likely to gather around the pore of the gel holder cassette. When I reprobed with GAPDH, I can see the GAPDH blot with these dots still.
I have performed some recombineering protocols and realised that the chances of my plasmid being in a multimeric state are quite high.
I previously designed 7 primer pairs that will produce alternating amplicons of 500 and 700 bp around my recombineered plasmid (which is 35kb) just so that I could get an idea that no weird recombination events occurred when looking at it in a gel.
Anyways, I did the 7 PCR reactions on a control with the original plasmid, and they produced the expected pattern, but when performing it on my miniprep-purified plasmid I was obtaining a lot of bands of all sorts of sizes (larger and shorter than expected amplicon). Funny thing is that these multiple bands seemed to follow the same pattern in all my replicates (different pattern for each primer of course, but same throughout the different colonies tested) which makes me rule out the possibility of salt contaminants affecting primer binding etc. I thought it might be bacterial genomic contamination that was being amplified, so I performed a CsCl-ethidium bromide density gradient to purify it and sent it off for sequencing.
But now Im wondering, would a multimeric plasmid yield multiple bands if amplified with a single pair of primers?
By the way, I can't run it on a gel to assess if it's multimeric because of its large size 35kb, although I am going to ask if anyone at my lab has a pulse field gel electrophoresis just in case.
Hello, I have developed a Surface Plasmon resonance sensor using LED of wavelength 635nm and CMOS webcam as source. I am using the diverging rays of the LED as the change in incident angle. When I put silver coated glass slide on the prism I get a dip at a particular angle. I have test the sensor by immobilizing with MUA , EDC/NHS and IgG. The sensor can detect the shift in angle for all the layers. But when I put liquid dielectric medium like DI water, BSA or PBS buffer the shift disappears. I can monitor real-time data with the webcam and so when the liquid sample is passed I should be able to detect the shift. I have attached the file of how the dip looks like.
As is well known, Continuous models are richer , more powerful and above all, more intuitive , easy to understand and extendable. So let us say, we are trying to find a biomarker for a disease \ trait. Instead of just looking at absence / presence or frequencies, could one try to establish continuous trackers / markers that positively or negatively correlate with the propensity to contract the disease , such as concentration levels of a chosen set of biomolecules ?? Possibly, this may involve some kind of preliminary pathway analysis. Could one also look at morphological parameters (fractal / scaling dimension of tumors etc ??) ??
Hi there, I have read that some (but not all) lentiviral transfer plasmids can be used in transient transfections to achieve transgene expression and those that can are primarily third-generation constructs. I would like to know if the pLVX vector is considered a third-generation construct and can be transiently transfected in cells.
We wish to set up a tissue culture lab but we are constrained by space available to us as a dept (Plant Science & Biotechnology)-We are trying to squeeze in all the lab units into the available laboratory space; tissue culture , molecular biology and plant pathology labs. We have designed the proposed lab with all the units in place taking all the safety precautions into consideration in the design. There is no issue with tissue culture and molecular biology labs all in place under the same roof but my worry is about including plant pathology lab even though it can be accommodated because of the problem of contamination. As the head of department i want to carry everybody along; the plant science ( for plant pathology) and the biotechnology ( for plant tissue culture+molecular biology) staff for logistic reason and otherwise. With a background in biotechnology this is the dilemma I am facing trying to squeeze in all the three lab units against my worry about contamination. Pls i need advise and suggestions from experts in the field of Plant tissue culture , molecular biology etc on this proposed multi-purpose lab. Attached is a sketch of the lab.
Some of my backbone expression vector are clearly contaminated by this IS. I use DH5alpha or TOP10 for routine molecular biology and quite surprise to observe this type of insertions in construction deriving from them...
We know that putting repetitive sequences in a single plasmid may initiate recombination in those plasmids. If recombination happens, the sequences in between those repetitive sequences may get excised out.
Can this be prevented by having the repetitive sequences in two separate plasmids (with two different backbones)? Would recombination still occur in between the separate plasmid systems?
P.s. I am working with a bacteria which is almost genotypically identical to its wild type variant. It is also not a recA- strain.
Aside from inhibiting protease activity (not effectively working), I read some papers about doing modifications to the antibodies:
using unnatural amino Acids
But, are there better options, more commercially applicable?
When staining with hematoxylin and eosin of a muscle biopsy from a patient with T341P desminopathy, we observe accumulations of inclusions similar to nuclei (arrows in figures 1 and 2, x280). And outside of these accumulations - adipose tissue, which used to be muscle tissue. There are no such massive accumulations of inclusions in adjacent muscle fibers. We assume that clusters of inclusions are not nuclei? Figure 2 is the inverted figure 1.
I am working with RNA switches for tuning few genes for production of metabolites in Lactococcus. I have observed recombination events in my plasmids when I culture the respective strains for 24hours in a bioreactor. I am hypothesizing that the high cell density (almost 10 OD after a 24 hours bioreactor culture) causes recombination in between the switch-trigger pair sequences in the plasmids. I have verified this after sequencing them.
The reason could be that these switch-trigger sequences form very strong secondary structures and have reverse complementarity to each other, and thus recombine and omit out the sequences in between.
Now these switches are essential to my work. So I can not leave them.
Can you recommend me regarding what genes I can knock out in Lactococcus lactis for preventing these recombinations?