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Molecular Biology - Science topic

PCR, Cloning, Restriction Digestion, Ligation, Transformation, Plasmid et al
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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.
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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.
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No, you cannot perform TaqMan assay using average regular use PCR master mix.
If you wish to perform PCR using the regular PCR mix, the assay is no longer called TaqMan Assay because the defining feature of a TaqMan Assay is the probe. This small piece of DNA matched to the DNA template being measured has two special molecules attached, a fluorescent reporter dye (R) and a quencher (Q). While both molecules are attached to the probe, the fluorescence of the dye is suppressed by the quencher. These probes bind to the template DNA after it has been denatured into single strands but before it has begun duplicating, making sure that all duplications of the template interact with the probe.
During the PCR reaction, Taq DNA polymerase extends the primer through the polymerase activity, as it approaches the probe it displaces the probe and cleaves it through the 5′ to 3′ exonuclease activity. This separates the reporter dye and the quencher dye from the probe, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected in “real-time” directly by monitoring the increase in fluorescence of the reporter dye with an automated PCR system.
The assay which you would wish to perform is called two-step reverse transcription-polymerase chain reaction. In this assay, two enzymes are used namely, reverse transcriptase to produce single-stranded cDNA copies, which are then used as templates in an amplification reaction catalyzed by a thermostable DNA polymerase. This assay is the traditional method of RT-PCR in which the two synthetic reactions are performed separately and sequentially.
The TaqMan Assay is a real-Time PCR assay which detects the accumulation of amplicon during the reaction. The data is then measured at the exponential phase of the PCR reaction. The assay which you may plan to perform using average regular use PCR master mix is a type of conventional PCR using agarose gel which is not as precise as qPCR. By using the regular use PCR master mix, you cannot perform qPCR because for qPCR one requires the fluorescent reporter molecule such as fluorescent dye, a labeled oligonucleotide primer or probe such as (TaqMan Probe) for fluorescent detection which is monitored by the automated PCR system. Real-Time PCR makes quantitation of DNA and RNA easier and more precise than conventional PCR.
So, if you wish to use the average regular use PCR master mix, you need to perform the two-step reverse transcription-polymerase chain reaction and not qPCR.
Best.
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Greetings, it is mentioned in your site that your research regarding cellular and molecular biology is also published (indexed) in pubmed, however i cannot seem to find it. could you please help me?
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doi: 10.7717/peerj.13489. eCollection 2022.. 2022 May 31;10:e13489.PeerJ
Prospective quantitative gene expression analysis of kallikrein-related peptidase KLK10 as a diagnostic biomarker for childhood acute lymphoblastic leukemia
Shwan Majid Ahmad 1, Basima Sadq Ahmed 2, Karzan Ghafur Khidhir 3, Heshu Sulaiman Rahman 4
Affiliations collapse
Affiliations
  • 1Department of Biochemistry, College of Medicine, University of Sulaimani, Sulaimaniyah, Iraq.
  • 2Department of Biochemistry & Clinical Chemistry, College of Pharmacy, University of Sulaimani, Sulaimaniyah, Iraq.
  • 3Department of Biology, College of Science, University of Sulaimani, Sulaimaniyah, Iraq.
  • 4Department of Physiology, College of Medicine, University of Sulaimani, Sulaimaniyah, Iraq.
  • PMID: 35669967
  • PMCID: PMC9165590
  • DOI: 10.7717/peerj.13489
This article is indexed in pubmed.
For more entries, you have to contact the publisher directly; there is no automatic indexing, with respect to RG.
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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 !
Philippe.
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Certainly, the length and sequence of the 5' untranslated region (5'UTR) can potentially influence alternative splicing, as changes in the 5'UTR can alter the accessibility of splicing regulatory elements, affect translation initiation through the Kozak sequence, and impact the secondary structure of the mRNA. Additionally, the use of a strong promoter like pCMV can lead to high gene expression levels, potentially influencing spliceosome assembly and kinetics of RNA processing. In your case, the observed splicing pattern in the pcDNA3.1 vector might be a consequence of the long 5'UTR and strong promoter, which could be introducing or disrupting splicing regulatory elements. Further experiments involving different promoters and 5'UTR modifications are necessary to elucidate the specific mechanisms underlying the observed splicing differences.
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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
Suggestions?
Thank you
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It will be classic but you mentioned using a 30-minute reaction time, consider extending it slightly, as sometimes longer incubation times can improve RPA results, especially when using DNA extracted from complex samples. While 400 nM is a typical primer concentration for RPA, you can try titrating the primer concentration to optimize the reaction. Sometimes, higher or lower concentrations may work better depending on your specific setup. While the primers worked well in PCR, they might not be optimized for RPA. You could consider redesigning the primers specifically for RPA conditions. RPA primers may have different requirements, such as longer lengths (usually 30-35 bases) and specific GC content. First things that come to my mind. Good luck.
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Is there any manual way to isolate recombinant fosmid DNA from E.coli cells in the absence of isolation Kit?
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Dear Farah, I am struggling with the same issue.. Could you please tell what worked for the isolation of your Fosmid DNA?
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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
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Dear Igor, here is what I could find:
original depositor: J. B. Waterbury << (I) CCAP << (I) Ernst-Moritz Arndt-University
Original name designator: Myxosarcina chroococcoides; Chroococcidiopsis thermalis,
Source! Soil sample, near Greifswald, East Germany
Ref: Komárek, 1972; Waterbury & Stanier, 1978; Rippka et al., 1979
PS: I am to answer your email very soon...
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We are focusing on Biotechnology, Food Technology, and Molecular Biology students.
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Since there is no thesis writing universally in undergraduate level probably it must have meant long essay type project writing !
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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.
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Do you wish to publish only in open-access journals? These days most journals offer a transformative publishing option, wherein you can choose to publish under a subscription model and you do not have to pay any money.
Elsevier, Springer, Taylor & Francis, Wiley- all these publishers have a hybrid mode of publication and you have to pay only if you choose to publish in an open-access model. These publishers also have a journal suggester/finder page where you just have to put your title, abstract, and keywords and you will be suggested relevant journals. There you can select the journal based on the scope of the journal and opt for the subscription model when submitting, so if in case the paper gets accepted then you don't have to pay at all.
Best!
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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.
Sincerely,
Zahraa Ozeer
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My Dear you apply for Canada visa and jobs along with resume.i can say that you can get there higher education with scholarship as you are scholar.
Very happy for your valuable open letter.i do not know in your united Arab Emirates citizenship.
Ok proceed.
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Molecular dynamics simulation , bioinformatics , molecular docking
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RAM= 32 GB or higher
Processor= Intel core i7 or higher
High-end GPU instead of CPU
Linux OS
I would suggest using a workstation instead of a laptop.
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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?
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Just to update, I've successfully transferred the small proteins / peptides from 20% acrylamide 1.5mm gel to a PVDF membrane using 250mA for 45 minutes. In case anyone googles this question :)
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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?
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With the progression of the detected case of desminopathy, the appearance of viruses and gram-negative rods was established, there was an excessive bacterial growth of the fecal microbiota with a pronounced increase in transient microorganisms, an increase in endotoxin. The results are presented in the article: https://www.researchgate.net/publication/372952519_CHANGE_CHARACTERISTICS_IN_SALIVA_AND_FECES_MICROBIOTA_OF_A_DESMINOPATHY_T341P_PATIENT
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I am looking for a PhD Position in Microbiology, Virology, and Molecular Biology.
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Bonjour,
Pour le moment tous nos projets sont pris par les doctorants.
Cordialement. M. SIDQUI
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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!
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Abhijeet Singh Thank you for your response and mentioning my earlier post! My belief is that researchers would know tools that are missing based on the fact that they would run into such problem often during their research. If there is some manual analysis task that researchers can automate, I believe that PeptiCloud can be the perfect platform to develop and make those tools publicly available. (For instance, PeptiCloud has a unique feature that allows users to further alter codon sequence of each amino acid after codon optimization with respect to a specific bacterial strain). With that being said, if you could check out PeptiCloud for yourself and see if anything could be added or improved, that would be greatly appreciated!
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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
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There could be several reasons why you're not achieving the same transformation efficiency as the paper. Here are a few things to consider: Quality of Plasmid DNA: Ensure that your plasmid DNA is of high quality, pure and not degraded. Use a reliable method to isolate and purify your DNA. Competency of Yeast Cells: The competency of yeast cells is critical. Make sure that the cells are appropriately prepared, not too old or too young, and handled gently during the preparation process. Plasmid DNA Concentration: The amount of DNA used can impact transformation efficiency. Verify the concentration of your plasmid DNA using a reliable method such as spectrophotometry or Qubit fluorometer. Transformation Protocol: Be sure you're following the protocol closely. Small details, like the temperature and timing of heat shocks or the voltage and cuvette gap in electroporation, can make a big difference. Media and Growth Conditions: The choice of media, pH, temperature, and even the type of agar can affect transformation efficiency. Strain Variation: Different yeast strains can have different transformation efficiencies, even with the same plasmid. Make sure your yeast strain is identical to that used in the paper. Experiment Reproducibility: Even when a protocol is followed precisely, achieving the exact same results can be challenging due to variability in conditions and materials.
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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
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Thanks Wolfgang, I appreciate the detailed answer!
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Yes unanimous agrregation of protein is cause of neurodegegenerative symptoms of molecular biology means bad disease of suffering like Parkinson,Alzheimer's etc .
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Yes, I agree with you that aggregation of protein is bad and is a cause of neurodegenerative diseases like Parkinson and Alzheimer's. Unfortunately, the mechanism explaining protein misfolding and protein aggregation, is not clearly understood at the molecular and cellular level.
It is well understood that functional proteins must pass through a quality control process in terms of folding to perform various physiological functions like catalysis, cellular transport, signal transmission and regulation, etc. However, there are a variety of structural and environmental factors that influence this process negatively. If you would be interested, I would like to list a few of them.
1. The protein structure, both the primary and secondary structures are most important for the physical and chemical characteristics. However, it could also be responsible for aggregation. For instance, the position and the number of hydrophobic amino acid residues in proteins may influence the aggregation behavior. The most common types of secondary structures, the α helix and the β pleated sheet may also have a role to play in protein aggregation.
2. Mutations play a determinative role in protein aggregation, and they may dramatically alter solubility, stability, and aggregation tendency of proteins. For instance, thermally stable proteins may change its stability even with a point mutation in its structure.
3. Post translational modification, especially phosphorylation plays a significant role in neurodegenerative diseases. For example, Alzheimer’s associated with tauopathy due to aggregation of the tau protein. In the brain, tau protein is found in neurons, and it can be phosphorylated with kinase enzyme. Thus, formation of aberrant tau aggregates accumulates in neurons, thereby exerting their toxic effects causing neuronal loss and synaptic alteration.
4. It is well-known that oxidative stress can cause protein oxidation, in particular, free radicals and ROS. Toxic free radicals can convert proteins into aggregation forms or proteins can be aggregated by conformational changes.
5. Environmental pH plays an important role in protein aggregation due to changes in net charge on protein. At low pH proteins may show aggregation tendency.
Best Wishes,
Malcolm Nobre
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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.
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Your analogy is very interesting, dear colleague.
Although the main cause of any form of myofibrillar myopathy is a violation of the structure of the protein components of sarcomeres caused by genetic mutations, why not assume that due to mutations, the sensitivity of the postsynaptic membrane of myofibrils in myofibrillar myopathy to acetylcholine may also be impaired.
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Hi everyone.
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?
Thank you.
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Philip G Penketh is correct.
The Dalton symbol Da, is also sometimes used as a unit of molar mass, with the definition 1 Da = 1 g/mol.
Therefore,
1Da= 1g/mol
1 kDa = 1000 g/mol
So, 18kDa = 18000g/mol
Now
18000 g ---- 1M ---- 1L
18g----------1M -----1ml
18000mg --- 1M ---- 1ml
The protein concentration is 0.6mg/ml.
Therefore,
0.6mg x 1M / 18000mg = 0.0000333M = 0.0333mM= 33.3μM.
So the protein concentration (in μM) as per the size (18kDa) and concentration (0.6mg/ml) is 33.3μM.
Best.
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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?
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I'd try isopropanol to remove the formamide. Should be more efficient than ethanol (at least from experience with precipitating nucleic acids from aqueous solutions). Basically, any solvent that is partially miscible with formamide should do the job, as long as the RNA doesn't dissolve in it. So, in the end, it will precipitate.
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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.
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Yes, antibodies for immunofluorescence of frozen sections and cells can be shared. Antibodies used in immunofluorescence experiments might be shared among researchers or colleagues in order to conduct comparable experiments or validate results. Sharing antibodies enables replication and validation of findings, fostering scientific transparency and collaboration in the research community.
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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.
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What I usually do is after activating PVDF membrane with methanol for 2-5 minutes, use Western-Blotting transfer buffer (usually is Tris/Glycine buffer containing 20% methanol) to wash the PVDF membrane for 5 minutes before do the transfer.
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If we take a gel picture (Image attached) then in lane 1 and 2, all the bands are Polymorphic bands? or they are monomorphic? 
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Thank you all for your answers.
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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
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Ammonium Sulphate precipitation works and may help resolve the particles by size. Note that the salt itself may affect the stability of the particles. In my hands, a 45% cut contained TMV CP monomer and multimers up to ~300k( analysed with boiled, reduced SDS-PAGE). I have read elsewhere that a 15% saturation is enough to precipitate rods.
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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.
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Animal testing You can contact us This is our area of expertise
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Hi everyone,
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 ?
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Philippe Paget-Bailly I believe that the switch from -80°C storage to -70°C storage for environmental reasons is a valid and sensible decision. The commonly used guideline of -80°C storage temperature originated from the technical limitations of cooling fluids used in the past, rather than being based on the long-term stability of samples.
The primary motivation for switching to -70°C storage is the significant reduction in energy consumption. It has been observed that the energy required to achieve and maintain temperatures lower than -70°C increases exponentially. By transitioning to -70°C, laboratories can potentially reduce energy costs by up to 30% compared to using -80°C freezers. This reduction in energy consumption aligns with the growing emphasis on sustainability and environmental responsibility.
The decision to switch temperature settings should be supported by scientific evidence and careful consideration of the specific samples being stored. The resources provided in the discussion include studies and information sheets that discuss the stability of various samples at different storage conditions. These resources can help researchers assess the impact of the temperature change on the integrity and longevity of their specific samples.
It is important to note that while the change in storage temperature may not directly affect the stability of the samples, researchers should ensure that the new storage temperature is within an acceptable range for maintaining sample quality and functionality. Factors such as the nature of the samples, intended duration of storage, and any specific temperature requirements should be taken into account.
In conclusion, the decision to switch from -80°C to -70°C storage for environmental reasons is a valid choice that can potentially reduce energy consumption without compromising the stability of samples. It is important for researchers to evaluate the specific requirements of their samples and consider the available scientific evidence when making such a transition.
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Hello,
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?
Thanks
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After earning your Ph.D. in biology, you may seek a postdoctoral job in computational biology if you meet the prerequisites for the position. Computational biologists come from a wide range of disciplines, including biology, genetics, biochemistry, and other relevant topics.
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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.
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Hello, I want to determine the TP53 and 13q14 status and telomere length in primary CLL samples, can you advise any reagents?
I select two reagents to define together: 1) https://metasystems-probes.com/en/probes/xl/d-5067-100-og/
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Target: SYBER
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Dear Soufiane Rabbaa
Add a non template control (NTC) in one PCR vial and test it.
A non template control is leaving the sample without cDNA. Usually NTC is used to check whether your cDNA is contaminated or to check primer-dimer formation. Here you can use the NTC to rule out which parameter (template quality, primer,reagents,dye) is to be rectified and troubleshooted.
All the best
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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.
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Yes, there are a few studies that have investigated the effects of centrifugation on RALA peptide vector complexes. In general, these studies have found that centrifugation can lead to aggregation of the complexes, which can result in larger particle sizes. This is likely because the centrifugal force causes the complexes to collide with each other, which can damage the complexes and cause them to aggregate.
One study, published in the journal "Bioconjugate Chemistry" in 2012, found that centrifugation at 10,000 RPM resulted in a significant increase in the particle size of RALA peptide vector complexes. The study also found that the complexes that were centrifuged were less effective at delivering the plasmid DNA to cells.
Another study, published in the journal "Molecular Pharmaceutics" in 2013, found that centrifugation at 10,000 RPM resulted in a decrease in the transfection efficiency of RALA peptide vector complexes. The study also found that the complexes that were centrifuged were more likely to aggregate.
These studies suggest that centrifugation can have negative effects on the properties of RALA peptide vector complexes. Therefore, it is generally recommended to avoid centrifuging these complexes unless absolutely necessary.
If you do need to centrifuge RALA peptide vector complexes, it is important to use a low centrifugation speed (e.g., 5,000 RPM) and a short centrifugation time (e.g., 5 minutes). You should also avoid resuspending the pellet after centrifugation.
It is also important to note that the effects of centrifugation on RALA peptide vector complexes may vary depending on the specific protocol that is used. Therefore, it is important to experiment with different centrifugation conditions to determine the optimal conditions for your specific application.
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It could include biochemical or molecular biology techniques.
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Thanks for all your inputs
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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
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Do you mean you are looking for a suitable journal for your work in this topic?
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Hello!
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?
Any suggestions?
Martin
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In my opinion, kit tubes should be fine. If adherence is the problem, you can short-spin it after adequate tap mixing.
Hope, it helps.
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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.
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Considering the necessity of growth factors for cells even for a few minutes, I usually use medium with fbs 2%, but serum-free medium is not recommended.
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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.
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Don't forget that the transcription start site might be very far away from the ORF, especially if the ORF is part of an operon. So don't just look immediately upstream from the ORF.
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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
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There could be several possible reasons why no bands are observed in the PCR amplification of 16S rRNA of E.coli genes:
  1. Contamination: Contamination with other organisms, DNA, or PCR reagents can lead to false-negative results. Make sure that your reagents are free from contamination and that you use appropriate precautions to avoid contamination during the entire PCR process.
  2. Annealing temperature: The annealing temperature of the primers may be too high or too low, which can lead to poor amplification or no amplification. You can try adjusting the annealing temperature to optimize the PCR conditions.
  3. Melting temperature: The melting temperature (Tm) of the primers may be too low or too high. If the Tm is too low, the primers may anneal nonspecifically to other regions of the DNA, resulting in no amplification or non-specific amplification. If the Tm is too high, the primers may not anneal to the template DNA, resulting in no amplification. You can try adjusting the Tm to optimize the PCR conditions.
  4. DNA extraction: Mistakes in the DNA extraction process can lead to poor DNA quality, low DNA concentration, or PCR inhibitors in the DNA samples, which can affect PCR amplification. Make sure that you follow a standardized DNA extraction protocol and use appropriate controls to monitor the quality and quantity of DNA samples.
  5. Genetic variation: There is genetic variation among different strains of E.coli, and the 16S rRNA gene sequences may vary among different isolates. Make sure that the primers you are using are appropriate for the E.coli strains you are working with.
  6. PCR conditions: Other PCR conditions, such as the extension time, cycle number, or buffer components, may also affect the PCR amplification. You can try optimizing the PCR conditions to improve the amplification efficiency.
These video playlists might be helpful to you:
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Hello,
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?
Thanks!
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The SV40 (Simian virus 40) promoter is a strong viral promoter commonly used for driving gene expression in various experimental systems. While the presence of a TATA box upstream of the transcription start site is a common feature in many promoters, the SV40 promoter is unique in that it lacks a canonical TATA box.
The SV40 promoter utilizes an alternative mechanism for transcription initiation called the "TATA-less" promoter. Instead of relying on a TATA box, it utilizes other elements and transcription factors to initiate transcription. The absence of a TATA box in the SV40 promoter does not necessarily impair its ability to drive gene expression.
Therefore, in your current cloning strategy where the transcription start site is located just 8bp from the end of the SV40 promoter, it is likely that the expression can still occur without the presence of a TATA box. The SV40 promoter contains other regulatory elements and transcription factor binding sites that can facilitate transcription initiation.
However, it's worth noting that the exact transcriptional activity may depend on the specific context and the downstream sequence elements present in your plasmid. Experimental verification, such as measuring the expression levels of your gene of interest, can help confirm the functionality of the modified SV40 promoter in your specific system.
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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!
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To perform a primary culture of non-parenchymal liver cells from mice, it is essential to choose the appropriate medium, determine the percentage of fetal bovine serum (FBS) to use, and decide on the necessary supplements. It is important to note that specific protocols may vary based on the intended application and the preferences of your laboratory.
For the medium, a commonly used choice for primary cell cultures is Dulbecco's Modified Eagle Medium (DMEM) or RPMI-1640. Both media are widely available and suitable for the growth of liver cells. The selection of the medium may depend on the specific requirements of your experiment or the protocols followed by your research group.
Regarding the percentage of FBS, a common range is between 5% to 10%. The choice of the exact percentage depends on the specific cell type and experimental conditions. It is advisable to optimize the FBS concentration based on the viability and growth characteristics of the non-parenchymal liver cells in your particular experiment.
As for supplements, commonly added components include L-glutamine, penicillin-streptomycin (antibiotics), and non-essential amino acids. The recommended concentration of L-glutamine is typically 2 mM, while the antibiotics are generally added at concentrations of 100 units/mL of penicillin and 100 μg/mL of streptomycin. Non-essential amino acids are often added at a final concentration of 1% or as specified by the supplier.
Additionally, considering the variability in experimental conditions, it is recommended to perform optimization experiments to ensure the optimal culture conditions for your non-parenchymal liver cells.
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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!!
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You've to be ble to analyse 3-D characteristics of both receptor & ligand and bond attractions which is very complex info, only known by very few highly trained chemists with high salaries and million usd bonuses work in pharma industries. But be sure almost nobody teaches these info to anyone since millions of usd gain, therefore you have to learn yourself if you are capable enough to learn !
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Hello,
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?
Thanks
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I should still work, because the water should be cool even the ice was melt overnight.
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"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." [1]
I add that the article mentioned that these discarded enzymes were targeting conserved sites in the study species.
[1]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?
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Yes, your reading is correct. The article implies that there is potential for future nucleotide variations within the conserved restriction sites that are located in variable regions of the 16S rDNA gene.
The possible mechanisms for such variations are mutations, insertions, deletions, or recombinations, which can occur spontaneously or as a result of exposure to environmental factors, such as UV radiation, chemicals, or antibiotics. These changes can accumulate over time and result in differences in the sequence and/or length of the conserved restriction sites, leading to the generation of different band patterns upon restriction digestion.
The time frame for such variations can vary depending on the bacterial species, its population size, its growth rate, and the selective pressures it faces. Some bacterial species have high mutation rates and/or frequent horizontal gene transfer events, which can result in rapid evolution and diversification. Others have lower mutation rates and/or stable environments, which can lead to slower evolution and conservation of certain traits. However, even slow evolution can accumulate changes over time, and it is difficult to predict the exact time frame for future nucleotide variations within conserved restriction sites.
Regarding your edited question, the frequency of new 16S rRNA gene variants in bacterial species can also vary depending on the factors mentioned above. Some bacterial species have high genetic diversity and high rates of recombination and horizontal gene transfer, leading to frequent emergence of new variants. Others have low genetic diversity and low rates of recombination and horizontal gene transfer, resulting in slower emergence of new variants. However, the 16S rRNA gene is generally considered to be a stable and conserved marker for bacterial identification and classification, and many conserved regions within this gene are used as targets for PCR amplification and sequencing.
These video playlists might be helpful to you:
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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?
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Hi Robert! Coming back in the past, which were your conditions?
Thanks!
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biofilm and quorum sensing genes of E.coli, not used drug but chemical material (Thiophene)
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Lack of aseptic techniques
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Hello everyone,
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.
Many thanks,
Tuba
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Bertrand Cornu Can Kiessling Audrys G. Pauža Mohamed Khashan Dino Santos Matias Thank u all. After many trials I have decided to use single temperature. I have to admit that still qPCR experiment is not so objective to me (changes according to conditions very easily). However, since everyone use the same method, and what matters is to compare the mrna level for the same protein, I guess it is fine.
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This would be similar in concept to using molecular biology’s BLAST algorithm.
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The Vienna Atomic Line Database (VALD) is a comprehensive database of atomic and molecular transition lines. It contains over 50 million lines for over 100,000 atoms and molecules.
The National Institute of Standards and Technology ASD (The NIST ASD) is a database of atomic and molecular spectral data. It contains over 10 million lines for over 80,000 atoms and molecules.
If the database contains lines that match the molecule's signature, then you will be able to identify the molecule in the spectrum.
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One example would be MBP and MBP-74, an interaction which can be disrupted by maltose when it binds MBP.
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Figure 1 shows Imatinib disrupts the protein-protein interactions of Bcr-Abl with some, but not all interaction partners.
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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.
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For cDNA synthesis, high-quality RNA with an A260/A280 ratio of 1.8 to 2.2 is ideal, and a minimum concentration of 1 μg/µL is usually recommended. For Real-Time PCR, the optimal RNA concentration is highly dependent on the gene of interest, and can range from 1 ng/µL to 500 ng/µL.
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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?
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The efficiency of Gibson cloning is higher than that of Golden Gate. Gibson cloning is a one-step assembly method that uses a DNA ligase enzyme to join two or more DNA fragments together. It is highly efficient, with reported success rates of up to 95%. Golden Gate cloning is a two-step assembly method that relies on a restriction enzyme and a DNA ligase to join two or more DNA fragments together. This method is less efficient, with reported success rates of up to 80%.
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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!
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I recently sequenced pHIT60 via https://www.plasmidsaurus.com. In case anyone is still looking, Iv'e attached the gbk file!
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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?
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No, introns are not considered in designing primers for full-length gene regions in the DNA template. Primers are designed to amplify only the exonic regions of the gene.
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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.
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Hello Hui Yee you could also try to use PICO to detect your MyoD. They claim to be much more sensitive compared to Co-IP/WesternBlot workflows. Here is an explanation of the technology:
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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.
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Arthur Kornberg, For the Love of Enzymes: The Odyssey of a Biochemist, is mainly focused on the discovery of enzymes involved in nucleic acid acid replication.
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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?
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Indumati Sharma Did you figure out the working temperature for this? I am going to perform this and am quite confused which temperature should I prefer? And can you please also specify the blank you used?
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Good day,
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.
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Agree that just because it's DNA-binding, that doesn't mean it can't be secreted too. But if you are sure that it is not secreted, necrosis-type cell is the only way I can think of for it to get into serum. Also, are you sure the protein is only expressed in monocytes?
As for your result, are you confident your protocol for making the cell lysate does enough to preserve protein stability? Cell lysates can be fickle in that regard.
Another potential issue are posttranslational modifications. If the secreted form has different PTMs than the intracellular one (which is common), this might affect antibody affinity.
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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.
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Andrew Paul McKenzie Pegman Hi! thanks for replying. I earlier read about mucilage and as I understand mucilage is the collective term used for the secretion by seed during germination. I am looking for specific term for these cells in image. These are single free floating round cells and these are viable even when they are away from the seed coat.
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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?
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Dear Sir you may try the protein separation with foam fractionation coloumn. it would suppose to help you
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What bioinformatics tools are available to help analyze and interpret large-scale molecular data generated from crop research?
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I highly recommend you to focus on your education and understanding the basics and fundamentals and not to spam here by posting questions and answering yourself.
Further, since you don't have the proper education to understand what software can be used for what it is totally illogical to talk about bioinformatics tools.
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How can you identify and isolate specific genes involved in crop yield or disease resistance?
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The answer to your question should be long. The disease resistance is usually conferred by a major gene while the crop yield is conferred by multiple minor QTLs/genes. Also, there are different approaches to isolating a gene of interest. There are several main steps to isolate the disease-resistance gene using a map-based approach.
1. Perform QTLs analysis using a small population like RILs, NILs, or DH that were derived from two parents carrying opposite genotypes.
2. Screen the plant carrying recombinants within the QLT of interest.
3. Develop internal markers to fragment the genetic window
4. Narrow down the genetic window based on the combination of genotype and phenotype of plants carrying recombinants.
5. Identify the list of candidate genes within the final genetic window
6. Validate the candidate genes to identify the actual gene conferring the trait of interest (using mutant analysis or gene transformation).
Good luck!
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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.
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I agree with both Gregory and Era. Try washing your sponges and transfer equipment thoroughly. I would also be careful with your membrane regarding containers etc you use for washing / staining.
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Hello,
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.
Thanks!
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Hi all,
Thank you for your answers,
I did a restriction digest with enzymes that cut multiple times and indeed, this plasmid has recombined in all sorts of ways except the one I was planning on...
I don't know if any of you have practiced recombineering before, but if you have I would really appreciate your advice regarding how to reduce unwanted recombination events in this type of cloning.
I am using an L-arabinose inducible plasmid for the λRed system. Are NEB10betas good cells for these protocols or maybe Stabl3 would be a better option? Also, would co-electroporating my plasmid at very low concentrations and the linear dsDNA into E. coli (which contains the induced λRed system-plasmid) help in avoiding these undesired recombinations?
Any other thoughts or help on how to avoid this?
Thanks!
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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.
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Did you find the solution? I am in the same trouble.
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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 ??) ??
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Difference equations can be useful for modeling discrete events in biological systems, such as the discrete stages of a cell cycle or the discrete events of a neural network firing. However, they may not be ideal for modeling continuous processes, such as the dynamics of a chemical reaction or the movement of a fluid through a network of vessels. In these cases, continuous models, such as differential equations or agent-based models, may provide a more accurate representation of the underlying biology.
One limitation of difference equations is that they assume that the changes in a system occur in discrete steps, and do not account for small changes that occur continuously over time. Additionally, they may not be able to capture the effects of small perturbations in the system, which can have a significant impact on the overall behaviour of the system.
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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.
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If your gene of interest is expressed via its own promoter (e.g. CMV, EF1a, etc) then you can always use the lentiviral plasmid in transient transfections, regardless of the lentiviral vector generation.
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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.
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Essien Archibong Okon I worked in commercial plant disease diagnostic lab under the same roof with Plant tissue lab from 2012 till 2021. Finally, we found such location incompatible with stable production of pathogen-free meristemic plants. Do not repeat the same mistake.
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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...
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Ho Okay, I've effectively seen it in the genome of DH5alpha but I did not find the genome of TOP10. Thanks a lot. In fact the insertion take place in the replication regulation area. May be it can influence the copy number to limit the toxicity of the cloned protein (a fluorescent protein). I will check on the website of Scarab Genomics to order a first batch of these clean E.coli ! Thanks again Alexandra !
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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.
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What origin of replication are you using? Rolling circle replication plasmids (especially with only a single stranded ori) like pWV01 tend to be much more unstable to secondary structure and short repeats than theta replicating origins such as pAMB1.
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Aside from inhibiting protease activity (not effectively working), I read some papers about doing modifications to the antibodies:
using unnatural amino Acids
using mPEG
But, are there better options, more commercially applicable?
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If you are talking about using the antibodies as the affinity matrix (such as in immunoaffinity chromatography), I would suggest that the way to avoid damage to the antibodies from proteases in a crude extract is to save the immunoaffinity chromatography for the last step in the purification, at which point the protease activity from the crude extract should be greatly reduced. Begin the purification with ion exchange, hydrophobic interaction, and/or gel filtration chromatographies. Use a protease inhibitor cocktail when making the crude extract.
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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.
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Dear Geir Bjorklund, Duc M. Hoang, John Hildyard, thank you very much for your answers and recommendations!
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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?
Thank you.
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To prevent recombination events in your Lactococcus lactis strains, you can consider knocking out genes involved in homologous recombination. The genes involved in the homologous recombination process in Lactococcus lactis include recA, recB, recC, and recD. By disrupting these genes, it is possible to prevent homologous recombination and reduce the likelihood of recombination events in your plasmids.
Additionally, you can also consider reducing the growth rate of the bacteria during your culture phase, which may help to reduce the rate of recombination events. This can be achieved by adjusting the temperature, pH, or nutrient conditions, or by modifying the composition of the growth medium to reduce the rate of bacterial growth.
Hope it helps