Questions related to Molecular Bacteriology
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... :)
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.
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?
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.
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.
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?
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.
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.
Fig 2 shows the genetics and Fig 3 shows the workflow. Section 3.5 shows the steps.
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!
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...
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
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).
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.
I tried using Normal LB and YPD still no success. I am conjugation using E.coli S17.1.
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.
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!
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
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?
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.
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.
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.
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.
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.
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......
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).
- 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?
The role of the infection with Brucella on the abortion of infected animals and no such occur in human being
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?
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!
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?
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 .
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).
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.
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.
i want to extract outer membrane protein of Pseudomonas aeruginosa and perform SDS page for their analysis
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?
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.
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.
This experiment would assess the potential of the bacteria for horizontal gene transfer.
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?
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.
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?
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?
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.
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!
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,
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.
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?
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.
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?
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 ...?
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?
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?
growing the bacteria(LB broth) for protein expression: the optical density(1.2) , is this good for protein expression(before IPTG).
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.
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
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?
Does someone have the protocol for chloramphenicol extraction from cultures of Streptomyces venezuelae?
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.
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...
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?
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.
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.
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
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!