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Is there any gliding bacteria (aerobes & anaerobes) isolated from the gut?
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Bacteria of the phyla Bacteroidetes are abundant in the human gut and oral microbiomes, and many members of this phyla have the ability to navigate surfaces via gliding motility. The Bacteroidetes represent about half of the bacterial population of the human gut microbiome. Kindly check the attached pdf.
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I want to use tetracycline as selection marker for my next experiments. I was advised to perform the experiments in dark. I want to know if this precaution is really required. I haven't found any literature supporting light-sensitivity of tetracycline. Please help...
Thanks in advance... :)
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Yes... while using the plates, keep lights in the vicinity off. During the incubation period no need to wrap the plates with aluminum foil. While storing the plates at 4C you must wrap the plates with the foil.
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Recently I'm having trouble with my maxiprep experiments and I don't know why. I'm doing exactly what I usually do: take a little bit of a glycerol stock to create a starter culture (5mL), let it grow during the day, and then around 6 pm I transfer the starter culture in 150mL LB (in a 500mL flask). Then the next morning I do the maxiprep. The main problems that I have are either: turbid supernatant after the first centrifugation to harvest the bacteria or the syringe gets blocked when I want to filtrate my lysate into the column. In the end, I have no DNA pellet or a really small amount of DNA. At first, I thought the problem was the glycerol stocks but even with new stocks, I encounter this issue. I'm thinking that maybe the cultures are overgrown?? If yes, why? That's how they do it in my lab and they never had an issue before. I'm using the Qiagen kit.
Thank you very much to whoever might help me.
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1. Check if you are purifying a low or a high copy number plasmid. If the former you might want to consider growing in a richer media than LB (e.g. TB) to increase the yield of plasmid per ml of culture.
2. Double check you are using the appropriate amount of antibiotics in your culture.
3. 5ml of culture to inoculate an 150ml ON culture would normally be excessive (e.g. 50-200 microlitres of an overnight is more common) unless you are using a bacterial strain that is a particular slow grower or the plasmid they carry makes the bacteria grow very slowly or your growth during the day is a very short time. You can measure the OD of the culture before doing the maxi-prep to make sure whether it is overgrown or not.
4. Are you introducing a clear neutralised lysate or could this be the problem?
5. A trick that works very well when there are problems with the elution is to pre-heat the elution solution before adding it to the column (e.g. to 50C). If your plasmid is very big, elution can be tricky and then pre-heating the elution buffer and letting in the column for a bit longer (5-10min) before recovery might solve the issue.
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I need to sequence an entire bacterial gene (6.1 kb). Is there a way to do it other than amplifying several smaller overlapping fragments for Sanger sequencing? Can I Sanger sequence the whole amplified gene using "walking primers", and if so, how is it techincally performed?
Is there any other method to sequence a single gene?
Thanks
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You can ,as you say, pcr amplify overlapping 800 base amplimers ( you get no useful sequence under the sequencing primer or for the next 30 bases). If you can amplify the whole 6KB in one amplimer then you just need overlapping sequencing primers at 800 base intervals along the whole sequence but just the one template dna. If you have a friend with a minion sequencer or NGS then new sequencing technology is good and quick but not cheap or easy to analyse the results
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Hi!
It is commonly used of silica membrae membrane in DNA extraction column to bind and release DNA. Yet I wonder can I simply use extraction tube to filter and collect ~20um beads (conjugated with DNA), then take out the whole membrane for the downstream reverse transcription, exonuclease and PCR. Will the membrane block or impede the enzyme to access the DNA on spheres? The membrane will be fully immersed in the reaction mix.
Thanks!
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Dear Friends,
Can anyone please inform, stepwise protocol to prepare Acidic phenol (pH 4.3.-4.5)? Am trying to isolate good Quality RNA from phenol rich tissue sample
Thank you
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Hello everyone,
I performed a classical extraction of the bacterial genomic DNA of four strains using Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v).
The 260/280 and 230/260 ratios are between 1.8 and 2. The concentrations vary between 600 and 1500 ng/µl (Attached file 2). A second dosage using Qubit fluorometer shows concentrations exceeding 600 ng/µl.
The gel shows distinct broad bands of high molecular weight (Attached file 2).
My questions are as follows:
1- Is it possible that my DNA is contaminated?
2- Is my DNA sample suitable for Nanopore Minion Sequencing?
Thank you for your help.
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Your sample concentration and ratio is good to go with any NGS platform (Even with Nanopore). Ratio of 260/230 should come near 2 but thats ideal and ratio will also work for You.
All the besst.
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I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other server that I can use?
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Dear Meydson Corrêa ,
Swiss-Model, Phyre2 or ModWeb work fine if your query protein has enough high homology to one or more templates in PDB. I-Tasser helps to identify more distantly related proteins (with similar fold). Raptor-X can also be helpful.
However, the most important questions here are "Are there any known templates for my query protein?" (and how good are they?), "How reliably can the region of interest be modeled?", etc.
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Once I get the bacmid I have a problem with the PCR. With the Negativ control without transposition I get a product of 300 pb. But with the PCR from bacmid with transposition (White colony) i can't obtain PCR product. I used am elongase and m13 primer with an especifc primer for my gene. I cant use only m13 primer forward and reverse because the product is too long. The pfast BAC that I employed is the dual yo clone two gens. 
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We never check the bacmid after transposition in bacteria; if your pfastback construct is OK then transform DH10bac, take white colonies (restreak them once on a new plate to be sure they are really white . then prepare bacmids from 2 or 3..different colonies and transform insect cells ... we only check baculovirus if we do not get the correct recombinant protein.
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I made a new bacterial strain with an unmarked deletion using 2 step homologous recombination (with sacB counterselection). The full protocol is given in the link at the bottom.
The protocol worked well and I thought I got my desired knockout strain. However, I have now found that I cannot electroporate plasmids into my new knockout strain successfully.
I don't think it's something unique about the gene I deleted, as I have another strain with the same gene interrupted by a transposon and it's fine (electroporations normal).
I've tried electroporations repeatedly with new reagents and another strain as a positive control. Every time, my old strains work (I get lots of electroporated colonies), but my new knockout strain gives no colonies. All the reagents and protocols work fine for my old strains.
Why could this be? I picked a second clone of the knockout and it behaves the same: No colonies from electroporations.
The KO strains seem to grow pretty normally in broth. I don't see a growth defect.
I used hygromycin selection to make the KO and hygromycin to select for electroporation transformants. Could that cause problems? The deletion mutant should have completely lost the resistance cassette, and, indeed, when I put my KO strain in hygromyicin selection it does not survive -- consistent with successful unmarked deletion.
Protocol:
Fig 2 shows the genetics and Fig 3 shows the workflow. Section 3.5 shows the steps.
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One possibility is that you have a contaminant strain and what you think is your KO is actually something else.
I would suggest designing PCR primers that will serve to diagnostically verify your final genotype construct is what you presume it is and also show that the material lost after homologous recombination is actually gone.
Are you electroplating a new replicating plasmid or are you relying on homologous recombination to see transformants? If the latter, it might be you have a deletion or rearrangement of the target region so the problem is the recombination event.
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Hey researchers!
So I was using a pET 101/D-TOPO (From Invitrogen) vector of old storage (5 years on -20 ºC) so the enzymatic efficiency of the TOPO has been reduced obviously, hoping it to work and definitely it did! But my question is why do I get smear bands during colony PCR in negative colonies because I have one positive colony as you can watch it in the attachments (More noticeable at the 3.jpg). The last two wells are positive controls followed by the negative control (where you can't see any smear band so it is not contamination).
What are your thoughts on this transformation? maybe bad ligation? inefficient process of ligation, enzymatic kinetics involved in a specific way for its old storage? plasmid instability?
Bonus: The non-specificities bands of the gene are from the own gene this could be because of the thermal program or something? because before cloning I purified my band from agarose gel with a kit so it would not be a problem to have those non-specificities
I'm anxious to read some answers!
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Are you using one plasmid primer and one insert primer?
I am wondering if ,in the absence of an insert, the plasmid primer is annealing and extending until the enzyme falls off giving random length smears but in the positive band case the reverse primer mops up the forward primer product giving a band but no smear
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I have read many articles where researchers get RVG-Exo by transfecting the exosome producing cell through RVG Plasmid. I wanted to ask if anybody knows, can we decorate exosome with RVG directly after isolation? Any reference please...
With thanks
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I haven't personally, but this paper did. Hope it helps!
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Commonly employed tests for detection of Streptococcus pyogenes include bacitracin sensitivity, PYR test and latex agglutination. But we can't completely rely on these tests as the results are not reproducible at times. With some isolates, I am facing the same problem, ie, when I repeat the tests for a second time the results are different especially with latex agglutination.How can I confirm the isolates as Streptococcus pyogenes. can we use spy1258 and dnaseB pcr for this purpose? how specific is these two pcr for detection of Streptococcus pyogenes. Please help me
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i agree with Dr. Godfred A. Menezes
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Fe-BABE conjugation can be used to find out the DNA binding region to the enzymes but Fe-BABE conjugation is a tedious process (mostly because of construction of single cysteine derivatives).
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I try to use the isolated colonies of Neisseria gonorrhoeae in chocolate agar for DNA extraction, but due to their clumping when I put them into liquid to make them dissolve, it perturb me for extracting DNA in a large collection of sample.
So how about the easiest way to doing that? Can I use the LB broth instead of fastidious or GCBL broth? What's the condition? Need I grow them in 5% CO2?
Thanks for your suggestion.
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N. gonorrohea is a fastidious bacterium, difficult to cultivate in liquid medium due to susceptibility to pH and divalent cation concentrations and a tendency to autolysis. Traditional broths (e.g. nutrient broth, tryptone soya broth, BRAIN HEART INFUSION) may allow limited multiplication of N. gonorrhoeae, but typically require inocula in excess of 10^5 CFU mL^−1 to achieve dense growth consistently
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I tried using Normal LB and YPD  still no success. I am conjugation using E.coli S17.1. 
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Yes, I know but things have changed a lot since :)
It's much easier now. I can send you the required tools if gene KO is what you are after...
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The pic given is the RNA isolated from Klebsiella pneumoniae using trizol reagent. Can any one please tell me what is the bulky band at the bottom? If it is sheared rna please suggest me a way to overcome it. Even trying with all precaution about rnase I am getting that band.
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See attached
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Free Journal For Microbiology
We have a Research about Bacteria staph...
Can anyone suggest us the name of a free journal publishing it?
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On the web page of Web of Knowledge (webofknowledge.com) you could find database of scientific journals, where you could sort them according field of interest, impact factor and other relevant metrics, that could help you chose right journal for your article.
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Hi, 
I'm looking for primers specific to Enterobacter cloacae complex or the entire Enterobacter species. The idea is to distinguish its members from other enterics by PCR. Does anyone know a sequence of such pair? 
I found multiple ready-to-use qPCR kits, but it's too advanced for what I need and there obviously is no info on the primers used. Can anyone help please? Thanks!
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I also need to quantify this bacteria, and the blow primer I found in this article https://patents.google.com/patent/CN102952881B/en
Hope it can help.
forward primer F: 5'-CATGACACCGGTGTTTCCCCAGT-3 '; reverse primer R: 5'-CGGTCGGTGAAGCCCAGAACCACTA-3'.
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Hi. I am trying to transfer RK2 plasmid from donor to recipient only. Does anybody give me an idea how I can distinguish transconjugants and recipients in the medium, they will have same genotype.Thanks
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You have most likely solved this problem, but for other reading, the simplest solution is to just Rifampicin/Naladixic Acid mark your recipient, then you can use either/both to select for recipient strain. You will need another selection marker for your donor DNA to select for transconjugants.
Just start at Rif 10, then build up to ~Rif 50 - 200, as to not allow spontaneous mutants of your donor strain.
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Unsuccessful experiment with ruthenium red
To distinguish between EPS-producing (S. thermophilus) and nonproducing (E. coli) cells, M17 agar medium containing 0.08% ruthenium red and ruthenium red milk plates (RRM) (consisted of 0.5% yeast extract, 10% skim milk powder, 1% sucrose, 1.5% agar and 0.08 g L- of ruthenium red) were used. After incubation at 37 ºC for 24 h and 48 h, every strain gave whitish (maybe pale pinkish) colonies. It was unclear.
Stock solution: 0.0877 g of ruthenium red [Ruthenium(III) chloride oxide, Alfa Aesar] in 8.77 ml distilled water was sterilized.
0.8 ml ruthenium red stock was added to 100 ml molten M17 and 100 ml molten RRM agar just prior to pouring it into petri plates. What could be wrong with that?
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I only had success myself using this method when I used Ruthenium Red from Sigma. I used one from Acros Organics originally and even though same formula, weight and CAS# received totally different results. Your agar should be pink to red. The plates should also be protected from light.
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Hello everyone,
I am hoping to observe adhesion microscopically using staining. I have been doing adhesion experiments on 96-well polystyrene plates but I want to be able to visually observe the attachment.
Has anybody successfully done this? What type of microscopic slide should I be looking for?
Thank you for your help.
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I have had communication with another researcher regarding the procedure, with which he checked with BCA.
For both of us, what worked was to bind commercial mucin to a NUNC maxisorp plate using acetate buffer (pH 5.0). A drying step was utilized after incubation with the mucin.
Take note that different approaches for mucin binding to the wells are different as the composition and concentration of mucin complexes used are different. But maybe the above steps may also be helpful for you, especially with the drying step.
Here is the link to our paper that details the approach we have used:
Best,
Valerie
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Why lactobacilli have intrinsic resistance toward Vancomycin?
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It's well-known that in several species of LAB, the terminal d-alanine residue is replaced by d-lactate or d-serine in the muramylpentapeptide, preventing vancomycin binding and therefore becoming resistant to this antibiotic.
Regards
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I want to study genetic diversity of Uropathogenic E. coli  .I  DNA extraction methods for E.coli by boiling.but i don't band in elektroforez.
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Dear Zeynab Faraji and everyone with the particular interest,
The RAPD is a very demanding technique, as it requires a lot of precision and the right PCR conditions to be maintained. Firstly, if the RAPD is failing, I would suggest to perform the gradient PCR. I had assessed RAPD with DNA extracted by boiling method and the results were really great, of course, after performing the gradient reaction and finding the best conditions.
In addition, another cheap, repeatable and more reliable method for the genetic diversity of UPEC is ERIC-PCR.
Good luck.
Rūta
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Sequencing of human genome at the center of interest in the biomedical field over the past several decades and is now leading toward an era of personalized medicine. During this time, DNA sequencing methods have evolved from the labor intensive slab gel electrophoresis, through automated multicapillary electrophoresis systems.
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Hi Mushtak,
it's simple but very reliable for small studies as we known. there is two types of electrophoresis, agarose gel electrophoresis, and the second used by old AB sequencers, polyacrylamide gel. for what you asked, the gel (POPn) is included in capillaries by trough your samples will migrate. since your samples (bands or sequences) are fluorescent (4 dyes are used), they will be detected by the CCD camera at end of electrophoresis and interpretation of sizes will be done.
fred
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I'm studying bacterial resisatance to antibiotics and I need to make site-directed mutagenesis on a specific gene. I want t know if there are labs with this experience in Egypt.
Kind regards.
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following
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From the methods I have come across, I haven't seen any that claim to be capable of purifying nucleic acid from bacterial, fungal and viral cells. Is there a commercially available method out there capable of this? Also, most methods seem to focus on blood. Are there any methods out there for sputum? Thanks for your help.
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Check this paper! It can give you some ideas.
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It is well known that genes play an important role in controlling all biological activity in our life. Most of these are functional can either in gene expression or in the synthesis if protein......
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There are 20-25000 protein coding genes in human and we are able to manage with fewer genes due to multitasking by these genes. Multitasking is facilitated by formation multiple transcripts from the same gene. Multiple transcripts are formed by alternate splicing, alternate promoters and alternate poly A sites.
In addition to protein coding genes there are hundreds of small RNA coding genes which are regulatory RNAs (example miRNAs)
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The diffence between the two is the sequence information that is encoded. The tracrRNA or trans-activating crRNA is made of up of a longer stretch of bases that are constant and provide the “stem loop” structure bound by the CRISPR nuclease . When these RNA components hybridize they form a guide RNA which “programmably” targets CRISPR nucleases to DNA sequences depending on the complementarity of the crRNA and the presence of other DNA features (PAM sequence recognized by the nuclease).
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tracrRNA, crRNA and sgRNA
Attached figure is a sgRNA (“s” for single, they’re the same thing). sgRNAs were artificially made by humans and don’t exist in nature.
This gRNA design is based off of the crRNAs and tracrRNAs which naturally exist in nature. Nucleotides 1–32 is the naturally-occuring crRNA. Nucleotides 37–100 is the naturally occuring tracrRNA. Briner et al. added a GAAA linker between the two pieces to make them one single RNA piece. The main purpose of this is to simplify the CRISPR system so you don’t have to express three things (i.e. Cas9, tracrRNA, and crRNA). Instead, you just need Cas9 and sgRNA). This simplification is important when you start dealing with CRISPR applications; the less moving parts the more efficient the system.
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- I mapped my whole genome against the reference strain, and found 100 bp gaps between all the contigs? Is it something to be worried about? How can I overcome this shortcoming in my sequence?
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Hello,
my impression is that these gaps of 100 bp (100 "N") were introduced artificially and do not say anything about the actual size of the gap. Some people insert 50 "N", other 100 "N". If it is always the same number it comes for sure from the assembly. The next consequence would be the the sequence before these "N" and the one after it correspond too individual contains and do not necessarily be next to each other in the genome. Saying this, PCR won't help, except if you want the go for all combinations. Moreover, since you do not know the size and complexity of the gaps, you may even not be able to PCR amplify the gap (e.g. of the gap is 10000 bp in reality).
Well, if I would be you, I would submit the genome sequence as it is but I would first remove all these stretches of "N" and generate separate contains.
BTW, could you please tell us how many of the N100 pieces do you have?
Best wishes,
Ralf
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The role of the infection with Brucella on the abortion of infected animals and no such occur in human being  
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Hi,
It is believed that brucellosis causes fewer spontaneous
abortions in humans than it does in animals because of
the absence of erythritol in thehumanplacentaandfetus
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Hi all
Bacterial family use same genes for colonization or every strain use different gene?
Have you information in details?
Can you suggest review articles?
I wanna check a population of bacteria that have been colonized or not.
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At least different from species to species. Furthermore there are fine tunings at the strain level.
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I would like to explore the PKS, NRPS and relevant gene diversity in the metagenomic actinobacterial DNA of the mangrove sediment samples to understand the secondary metabolite production pattern. Can some one recommend / provide a simple and efficient protocol to amplify, clone and sequence the PKS, NRPS and relevant gene in the metagenomic DNA pool; specific to actinobacteria?
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Already specific primers are available to screes PKS and NRPS gene from actinobacteria. You can follow that protocol.......
Comprehensive Investigation of Marine Actinobacteria Associated with the Sponge Halichondria panicea
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Hi everyone,
I recently faced the trouble with bacterial contamination in cell culture, and it is not just as simple as discard all current cells and buying a new stock as many people suggest.
In my case, I have been using antibiotics cocktails, renewing all reagents, but the bacteria could not be removed.
I am now considering the centrifugation, but I wonder which centrifugation speed and time could be suitable (I read somewhere that 2000x g for 10 minutes can help to separate bacteria from cells, but I am afraid this speed and time could be harmful to the cells for the next culture).
And I am also thinking of using filter to separate my cells from bacteria, but I wonder if this is possible, and if so, what should be detail protocol for this?
Please anyone could give me some advice for my trouble.
Thank you so much for all your helps!
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I would strongly advise against using "decontaminated" cell lines. The best cell culture technique uses no antibiotics (or antimycotics) because if your technique fails, the infection is a warning; analogous to pain in a sprained ankle or knee. You want to know about the infection (injury). Even if you were to succeed in completely eliminating the infection (low probability), you might still have a mycoplasma infection as well. This would change the behavior of your cells and make future experiments unrepeatable by other labs, and probably even your own. Get your aseptic lab and technique in order, then keep your stock cells in antibiotic-free culture conditions. Of course, you should have a stock frozen and maintained in liquid nitrogen, so that you can replace your growing stock every 3 to six months (depending on cell doubling times). This protects against genetic drift of your stock cultures and will enable you to repeat experiments decades later and still get the same results.
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Hello Everyone, 
I am developing a system with two plasmids. One of the plasmids have been assigned the incompatibility group of IncH, the other is plasmid pK19mobsacB in which I am looking into the group. 
Assuming they have different incompatibility groups, would it be still proper or necessary for publication for me to check this? Keep in mind that I did insert a gene into pK19mobsacB, but it shouldn't effect its replication pattern.
If I can not assume  that the two plasmids are compatible based off of the groups they have been assigned to, what would be the best way of testing their compatibility?
Thanks.   
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Plasmid incompatibility is typically only a problem when you need the two plasmids with the same group maintained stably over multiple growth cycles without antibiotic selection. It's typically not an instantaneous thing. 
By the way, pK19, which I believe is the backbone plasmid, seems to have a pBR322 origin. That would make it a ColE1 plasmid. I can't remember which incompatibility group that is. Source: https://www.addgene.org/vector-database/3302/
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After repeatedly sub-culturing some of my strains of Streptomycetes, I find that their antimicrobial potential is diminishing. Are there some good methods for re-awakening their antimicrobial synthesis ? Currently I am trying many different types of media .
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Stocks maintained in glycerol 18% at -80 C
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It should be able to grow and induced in E. coli. The molecule of induction should not be IPTG ( It can be acetamide, Tetracycline etc). It should not have the following origins of replications (p15A, colA, pBR322, f1). 
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Hi,
One may try pCDF expression vector. It has CDF origin and is streptomycin resistant. 
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I am trying to isolate outer-membrane vesicles from Salmonella Typhimurium using ultracentrifuge method. My protocol is as follows:
1.      The Salmonella Typhimurium is grown upto end of logarithmic phase in 200 ml Luria Bertani Broth (pH: 7.2) at 37ºC.
2.      The culture is centrifuged at 10,000×g for 10 minutes at 4ºC.
3.      The supernatant is ultra-centrifuged at 1,24,000×g for 4 hours.
4.      I should get the pellet at the end of ultra-centrifugation. But I do not get the pellet. Sometimes I get a minute dot like pellet that is very less to do any type of analysis work.
Please guide me in this regard.
Keywords: outer-membrane vesicles, OMV, Salmonella, Typhimurium.
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Concentrate your supernatant from 200 ml down to 2 ml using Amicon Centrifugal Filters. You might then be able to see it. There are also plenty of published protocols for Salmonella OMVs isolation. Thanks
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Along with which virulence factors produced during that biofilm transition stage from the planktonic cells. Its good that it was found that cells experience shear stress and flow for formation of biofilms. 
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Time of forming biofilm or quality of that depends on many factor, especially virulence genes. In my project toxin-antitoxin, I had many strains that isolated from patient but only some few one had ability to producing biofilm.
Good luck with your research.
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i want to extract outer membrane protein of Pseudomonas aeruginosa and perform SDS page for their analysis
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thank you all for you kind suggestion 
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What is the role of extracellular DNA in bacterial antibiotic resistance and how can it be detected in the lab?
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Understanding the mechanisms by which biofilm bacteria develop resistance to antibiotics is paramount to expanding the treatment options available to patients with chronic biofilm infections. ... Extracellular DNA, a known component of biofilms, was found to induce antibiotic resistance
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Lipopeptides like daptomycin, surfactin, polymyxin, etc. have antimicrobial properties. They bind to the bacterial cells and creates pores in their cell membrane which results in cell lysis. What part or residue of bacterial cell do these lipopeptides bind?
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AMPs are not ideal drugs: they are far larger than typical druglike molecules, tend to be expensive to synthesize, and are prone to peptidase degradation when used internally . This suggests that the best case for a drug based on a natural linear AMP is a topical antibiotic, although daptomycin, which, (even though it is somewhat toxic to human cells), has been approved by the Food and Drug Administration (FDA) for therapeutic application in systemic and life-threatening infections caused by Gram-positive bacteria.
Several models have been proposed to explain the molecular mechanism of AMPs,(antimicrobial peptides) such as barrel-stave model  or More recently explained by both  Makovitzki and his colleagues , and  Vallon-Eberhard and his colleagues.
You can review the following :
Makovitzki, A., D. Avrahami, and Y. Shai. 2006. Ultrashort antibacterial
and antifungal lipopeptides. Proc. Natl. Acad. Sci. USA.
Vallon-Eberhard, A., A. Makovitzki, ., Y. Shai. 2008. Efficient clearance of Aspergillus fumigatus in murine lungs by an ultrashort antimicrobial lipopeptide, palmitoyl-lys-ala-DAla-lys. Antimicrob. Agents  Chemother. 
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Hello everyone, I am trying to determine Catalase activity (KatA) in B. subtilis from the exponential phase, cells were exposed to different concentrations of H2O2. However, when I determined catalase activity in the control and induced cells I see differences in the activity. How can this be interpreted just considering that I am working just with KatA? Considering that Catalases generally, being catalytically active enzymes do not follow Michaelis–Menten and follows Bonnichsen-Chance-Theorell (BCT) mechanism.
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The problem it´s that in the system it´s already overexpressed, I am comparing it in the same OD600, protein has been quantified, and actually when bacteria is exposed to H2O2, population starts decreasing
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Dear all, I am trying to estimate the relative amount of two bacterial strains in a consortium using qPCR with SYBYR Green and 16S primers specific to each of the strains.
So far I was able to design the primers, confirm that they are specific and quantify my bacteria.
The problem I have now is that my qPCR results (copies/ml) are overestimating my cell counts (CFU/ml). One of the strains has 4 copies of the 16S and even when divide my copy number by 4 I still have 10x (1 log) more copies than CFU.
The bacteria were collected in the early and late stationary phase.
Is this normal? I couldn't find any answers in the literature.
Thank you for your time.
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I think it depends on many conditions as16s rRNA copy numbers fluctuates during different growth phase, effect of different growth conditions. It is always suggested to use single copy gene because of variation in 16s rRNA copy numbers and high sequence similarity between rRNA gene so most of the times primers from the rRNA gene shows false positive results even in the absence of DNA template. According to my experience I suggest use single copy gene per genome that can specifically amplify your gene and also consider that qPCR can amplify live and dead bacteria and difference in bacterial culture conditions. For references about different approaches on estimating genome copy numbers please have look on this reference that might be useful for your study.
Jaai Kim a, Juntaek Lim b, Changsoo Lee.  Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: Applications and considerations . Biotechnology Advances xxx (2013) 
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May I have your opinion on the use of IGRA , especially T-spot on LTBI? I have personally experience the lack of reproducibility of T-spot, a first test being borderline positive and the repeated one negative, with only one spot. When a test is so inconsistent, even if it being very specific and sensitive, can we still use it in confidence to diagnose LTBI?
Hypothetically,
a) If a HCW, healthy with low risk of TB infection has a first positive test for healthy employment screening, will you treat them for LTBI once active TB is excluded? or will you repeat the test? what if it comes back borderline, or negative?
b) And if a patient, healthy with low risk of TB infection has a borderline test, does a second negative or positive test mean anything much?
What are your usual practise? Thank you in advance for the opinions.
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Thanks a lot Kuan
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I am exploring options to preserve bacteria namely, Staph. aureus, E. coli, P. aeruginosa and Salmonella typhi. I want to be able to preserve them for a period of 1 year and will be using them for genetic analysis.
Two methods seems feasible in my laboratory - one using Glycerol and the other using Paraffin Oil.
Which method is more preferable based on efficacy and labor requirement?
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 I prefer  Paraffin Method  
This is a simple and most economical method of maintaining pure cultures of bacteria and fungi. In this method, sterile liquid paraffin is poured over the slant (slope) of culture and stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture can be preserved form months to years (varies with species).
The advantage of this method is that we can remove some of the growth under the oil with a transfer needle, inoculate a fresh medium, and still preserve the original culture. The simplicity of the method makes it attractive, but changes in the characteristics of a strain can still occur.
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m
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The question is not clear
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I am looking at alternative options for quantifying bacterial viability in addition to colony plate counting.  I have a mixed species bacterial culture that may also contain epithelial cells.  I have tried ATP as a metabolic measure. The challenge I have is correctly quantifying bacteria ATP vs epithelial.  Is there a way to filter or eliminate everything but the bacteria?  How can I make sure I read only the bacterial ATP?  
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What is IZT assay?   
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Any idea? 
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These articles may be useful to you:
1- . Kilvington, S., and J. Beeching. 1995. Development of a PCR for identification of Naegleria fowleri from the environment. Appl. Environ. Microbiol. 61:3764-3767.
2-Pelandakis, M., S. Serre, and P. Pernin. 2000. Analysis of the 5.8S rRNA gene and the internal transcribed rs in Naegleria spp. and in N. fowleri. J. Eukaryot. Microbiol. 47:116-121.
3- Reveiller, F. L., P. A. Cabanes, and F. Marciano-Cabral. 2002. Development of a nested PCR assay to detect the pathogenic free-living amoeba Naegleria fowleri. Parasitol. Res. 88:443-450.
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My colleague and I have been attempting to integrate a recombinant CRIM plasmid into the chromosome of E. Coli Nissle 1917. As far as we know, we have been following a suitable protocol as suggested in the paper by Hardimann and Wanner (2001). However, we have not even been able to transform a single colony despite working on the transformation for several days. Does anyone have some advice for us, perhaps regarding the inherent nature of ECN and CRIM plasmids? Thank you very much, and any help is much appreciated.
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Have you tried electroporation? the growth phase of bacteria can also affect transformation efficiency, so I guess you can try it out with different setting of voltage and growth phase ( mid log phase works fine for me)
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Hello, 
To calculate E.coli cfu/ml, I usually calculate OD600 and according to that reading, I can find out cfu/ml for E.coli.
 Now I want to work with two extra bacteria: S. Aureus & S. Pneumoniae, Can I use the same technique to calculate cfu/ml?  or should I use another way because those two types of bacteria might have different shapes and densities! 
Thank you
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Hello Saud Alyafei. It will be best to make serial dilutions of your suspension (1/100 - 1/10,000), and plate an amount (10-100 ul) on agar plates. The next day, you can literally count the colonies per plate, and use a formula of CFU counting, taken into consideration of the dilution factor and the volume of suspensions. This will be more accurate compared to absorbance readings, since OD600 will also read the non-bacterial contents of your suspension, eg. waste materials.
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I want to identify, separate and quantify S. aureus and P. aeruginosa from dual-species cultures both from planktonic or biofilm. Could there be any other method to do so without using flow cytometry or fluorescence proteins?
Thank you in advance,
Legesse
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Thank you so much Lekieh Peekate. I am eager to read your work in this regard.
Have a nice time,
Legesse
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Hi,
 I am going to prepare Genomic DNA library for NGS, I have cultured gut bacteria on various media and scrap the plates,I use tiangen kit to isolate DNA but it is shared and for Genomic DNA library it can,t work, my Question is what can be the best protocol to isolate DNA, The culture plates contains almost 700-1000 bacterial colony of both gram positive and gram negative bacteria.
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 Thank you Marcela Krutova.
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Hi...
I have done 16 S rRNA sequencing of some of my bacterial isolates and one of my isolates is showing only 90% sequence similarity with reference strain as the maximum identity. Should I perform biochemical tests or is there some other way to identify the genus and species of this bacterial isolate?
Thank You...
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Thank u very much for sharing this valuable information.
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Greetings fellow microbial researchers, do you prefer to use Greenegenes or SILVA as a reference database for bacterial (16S) microbiome studies? Why? One obvious advantage of SILVA is that it has been updated fairly recently, whereas Greengenes hasn't been updated since 2013, but other than that, I'm not sure which is better.
I ran both on my data and compared the results (just the OTUs, not any actually community comparisons), and at the genus and family levels, SILVA annotates almost twice as many OTUs as Greengenes. At the class level, however, Grenngenes annotates nearly 100 more OTUs than SILVA. So for my data set, it is not clear which database will do a better job. Also, when both databases annotate a given OTU, they frequently disagree, and it's not clear to me which database in more reliable.
Thanks for the help.
P.S. these are Illumina MiSeq data.
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Dear Donald,
Choosing a database for bacterial 16S analysis is a vast discussion.
Personally, I would recommend SILVA for several points:
First, the database is maintained and updated regularly and second, the sequences are quality checked. Meaning that when you blast you have a more accurate result and you can be sure that the sequences (and so your alignment) are reliable. You can check directly on the Silva website for more informations (https://www.arb-silva.de/documentation/). After I do not really know Greengenes but if it was not updated since 2013 I would not rely on it.
Concerning RDP, although the database is updated regularly, it is more suitable for taxonomy analysis than metaprofiling. I did not used it in a while so you would need to have a look at how they are building their database but from my personal experience I have also noticed that the annotation was not that accurate at the OTU level as it is mostly based on NCBI data sequences which are not always reliable. That said it is a really good database for fungal data.
After it is all a matter of choice, every database has its pros and cons, it also depends if you want to compare your data against some other studies, therefore it is better to use the same database. SILVA is usually a reference among microbiological researchers, especially for bacterial 16S analysis.
Hope this helps,
Regards
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Most of the study methods I read were using CFU to determine the quantity of bacteria for the studies (mostly due to the fact that OD can't distinguish dead or live bacteria). It makes sense if the experiment is about cytotoxicity, antibiotic screening etc. What if I am interested in comparing different bacteria challenged cytokine induction profiles? Since dead bacteria particle could also induce cytokine release, would the CFU based method create a bias?
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It very much depends upon the type of experiment you are doing to ascertain which makes the most sense to use. However one major downside of CFU determination is that you only know the number post-facto, ie the next day. So it can be hard to use CFU during an experiment, rather it just tells you how many colonies you had afterwards. 
It might be easiest to determine an OD curve with CFU and possibly counting chamber analysis to ascertain the cell numbers at any given OD for your strain. Then you can use the OD value for quick measurements but correlate it to either CFU or total cell number based on your curve.
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I need a pair of primers which have both specificity and sensitivity for determining the number of P.aeroginosa in water (including VBNC), and a constant expression of reference genes in virulence measuring. I mean a primer from housekeeping genes (with a constant expression in any situation) wich is 100% specific and sensitive. Do you recommend Oprl, 16SrRNA, or ...?
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you can visit our college we can help you by staff of zoonosis diseases
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brucella, rpoB gene
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Dear  Khetam
Comparison of the rpoB nucleotide sequence of all Brucella strains  revealed specific nucleotide variations associated with different Brucella species and biovars,the rpoB gene polymorphism can be used to identify all Brucella species and most of the biovars, offering an improvement over conventional typing methods.
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Suppose I am using MacConkey broth for E. coli testing (MPN method) and after completion of test, observed results are negative for the presence of E. coli
Now the colour of medium will remain unchanged even after few days, so can we use this media for testing of E. coli of another sample .
Can we do this with any other media?
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Microbial culture media can not be generally used for microbiological tests more than once.
In this case, because:
The positive result in MacConkey broth for E. coli testing (MPN method) is acid+gas from lactose sugar fermentation.
The negative tubes from the first use do not contain positive lactose fermenting microbial types, but may be, they contain other microbial types cells (negative lactose fermentation), which when be used again, get contain two microbial types, both of them are negative lactose fermentation, but they have synergistic effect:
Which means, one of microbial type make metabolism of the culture medium ingredients and give new compounds (its metabolic products, not acid+gas), then another microbial type uses the metabolic products of the first microbial type and give acid+gas (but really it is false positive result).
So, for microbiological tests of samples, positive tubes have to be followed by recommendation tests (minimum Gram stained slide) to be sure that. positive result gets from the intended microbial type.      
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Could anyone please tell me where I can get the following strains?
AHL degrading strain Pseudomonas fluorescens (P3/pME6863)
AHL non-degrading strain Pseudomonas fluorescens (P3/pME6000)
Agrobacterium tumefaciens AT 136 (pZLR4)
Maybe someone could provide me with a sample?
Thanks :)
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my dear, 
i sorry, I suggest you to contact the people who worked with these bacteria.
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Can any one explain what is the difference of using 3 tube method instead of 5 tube mpn method. 
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Hi
you used 5 tubes for  lower error in method 
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growing the bacteria(LB broth) for protein expression:  the optical density(1.2) , is this good for protein expression(before IPTG).
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I definitely agree with Truc here, you want to find the right balance between the number of cells and the amount of protein yield you get at the end after IPTG induction. You can sometimes get a higher protein yield when induced at a slightly lower OD. It all just requires some optimisation. 
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So I have been carrying out a bacteria killing ability assay, which leaves me with proportional data for samples (i.e. the proportion of bacteria killed by an individuals plasma). One of the batches I ran had a large amount of variation (which is expected, samples ranged from 0.1-0.9 with a mean of 0.7), however, the second batch had a lower amount of variation and much higher mean (samples ranged from 0.75-0.99, with a mean of 0.96). I carried out a leveneTest in R and the variances are significantly different. Does anyone have any idea on the best way to deal with this problem. Each batch contained different samples. 
I did carry out the bacteria killing assay again for the second batch, however, plasma tends to lose its killing ability when going through freeze thaw cycles. This proved to be the case as in the second run for batch two, the variance was higher but the mean dropped way down to 0.23. I have tried log and square root transformations, but there are still differences in variance between the two batches. 
Not sure what I should try next, does anyone have any suggestions on how I should proceed? I am using R for all of my statistical analysis. 
E
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It dependes a lot on what are your specific goals on this study. With more information it would get easier to see which model could be applied to your case.
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Hi everyone!
Could anybody please suggest me, where to get CH184 E.coli strain from? This strain is streptomycin-pseudo-dependent strain.
If somebody has it, would it be possible  to kindly provide me the strain. It would be a great help.
Thank you in advance
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Is Escherichia coli and Klebsiella pneumoniae closely related? Do both of them produce green sheen color on EMB agar? A strain doubted to be E. coli identified as K. pneumoniae even after performing all the biochemical tests. Complete genome comparison of identified strain shows 99% identity with E. coli but in BLAST results it shows as K. pneumoniae with no listed E. coli as identity. Any literature to support this?
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Yes there's confusion between tests too. Green sheen colour produces on EMB agar (which indicates E. coli) but it is identified as Klebseilla. Also another observation is that green sheen colour changes to pink purple colonies after some time in refrigerator. Sometimes mix colonies form.
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Does someone have the protocol for chloramphenicol extraction from cultures of Streptomyces venezuelae? 
Thanks.
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I study with a Salmonella strain. This strain has plasmid which carries amp resistance gene. And i want to get the strain free of plasmid. 
How to get it? any ideas?
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Salmonella grows very well at 42ªC so it could be difficult to get rid of plasmids by only doing this. Be prepared for a lot of replicas.
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Bactera (Bacillus subtilis or Lactobacillus sp.) have levanses for use and production of levan. Levan is polysaccharide which may be present in the gastrointestinal tract as a prebiotic. I am curious if levan is carbon source for Saccharomyces cerevisiae and S. boulardii.
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Sacch. boulardii and Sacch. cerevisiae have many enzymes. It do sugar analysis as levanse 
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We have recently isolated a strain, which is a gram variant strain showing mucoidal colony morphology on nutrient agar plates and which glows (luminescence/ fluorescence) when viewed under UV light. The fluorescence was remarkably found around the colonies,  when centrifuged the property was found to be in the supernatant. IMViC results were like -+++, non- lactose fermenting, and showed acid + gas on sugar like Glucose, Mannitol and Sucrose, urease positive and citrate positive. 
Someone please suggest what could be the organism, we have given it for sequencing, still curious to know what it could be... 
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Probably Pseudomonas spp.  Do the oxidase test.  Pseudomonas spp. are oxidase positive.  How old was the culture when you did the gram stain.  Pseudomonads are gram negative small rods.  Do gram stain from a young culture, preferably on rich medium such as trypticase agar or Muller Hinton agar.
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Is it possible that A. tumefaciens A136 encounter some type of Mutation after serial culturing? if not then why it's not giving positive result even for positive control in Quorum sensing investigation?
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@Sergey Dobretsov, Sir I am using Marin Agar on which both A136 and my test strains grows well but not responding to X-Gal even to positive control. sir my protocols are as follow
1. Culturing of Reporter strains using Marine Agar.
2. Culturing of Positive and test strains > incubating them for 24/48 hours > culturing them with repoter strain A136 in T manner> incubatin them for 24/48 Hours at 28 °C > adding X-Gal > Cheking for QS activity after 12 hours then after 24 hours then after 36 hours then after 48 hours.
Sir is there any thing wrong in this protocol? if yes kindly guied me.
Respectful Regards,
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I'm trying to compare my MLST of Acinetobacter baumannii with the Eburst database, but I found that in the Eburst database, they don't have any database for A.baumannii so I decide to download the reference MLST (database) from MLST (Pasteur) profiles in http://pubmlst.org/ and make them into .txt file as attached in this post. Anyway, I can't compare because it show the 500 - Internal server error and don't know how to fix it.
Have anyone encounter this problem? How I can fix it?
Thanks a lot.
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Dear Natakorn, I had the same problem today. First I did in another browser (I changed from Chrome to IE) and I did in my house because the site was blocked in my institution. Indeed, my file does not have the headers and the species name. Only the ST and the alleles. 
Hope you had got it!
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Hi,
Is there a method to extract RNA of both gram positive and gram negative bacteria in their dual-species culture? I want to perform transcriptomic analysis, so I need RNA sample with high quality and quantity. I have tried method for Gram positive bacteria to extract RNA of gram negative bacteria, then it cause degradation. 
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I am working on the role of endophtic bacteria in plants during stress.
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Thanks
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I need to know the purification quality of these kits
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If you are worried about the purity of your sample, there are commercial products available to remove contaminating human DNA and significantly amplify signal:  https://www.researchgate.net/publication/298064100_Broad-Range_Microbial_DNA_Isolation_from_Clinical_Specimens_for_Universal_PCR_Diagnosis
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Hi, 
Anybody have the data of pathogenic and nonpathogenic strains list of staphylococcus aureus species? If anybody has pls respond.
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Staphylococcus aureus in itself is a pathogen, though a facultative pathogen, meaning there is no non-pathogenic strain of it.
Whether or not a disease develops is a matter of host-microbe relationship (certain host factors contribute to disease susceptibility while virulence factors drive the ability to cause disease). About 20% - 30% of healthy people carry S. aureus, in a few carriage may turn into disease. The extent to which a certain S. aureus may cause disease depends again on host-pathogen relationship and on the particular virulence of an isolate. Virulence is a trait of an isolate or strain, different from pathogenicity, which is an attribute of a species. Since in your topics you name also resistance patterns: antimicrobial resistance hampers therapy by narrowing treatment options but is usually not a determinant of pathogenicity and/or virulence.
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I'm isolating P. aeruginosa from an oil spilled soil using Mineral salt medium, thereafter go on to isolate the PCA from the organism but am not getting positive result, which other way should I go about that? I also want to know know the chemosynthetic way to produce this compound, PCA antibiotics. Thanks
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Thanks a lot, Marielsa Gil. I found your recommendation and directions very helpful. I Will get back to you on the progress soon. Thanks once more.
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I am looking for an agar medium suitable to S. thermophilus growth but not for other Sptreptococci. I know about:
ST medium (other strep would grown nicely maybe I could change the carbon source from sucrose to lactose)
M17+lactose: for sure other strep would grow
Salivarium mitis agar: enterocci and streptococci would grow here too
Thanks in advance! 
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