Molecular Microbiology - Science topic

Mercedes Vazquez Can I as you what plasmid you are talking about? Is it pCP20?

Many thanks?

Hi Abhishek,

Did you get it to work? I am using the transposome kit and the pir-116 cells now. I had no luck so far, but the control DNA works in the cells though. Do you have any recommendations?

You can try annotating your genome with tools like PROKKA, PGAP or RAST. And look at the predicted genes and other features

What ive seen is that in rare cases after an autoclaved high conc PBS (phosphate-buffered saline) solution has been stored cold, it can result in precipitation which may create the impression of contamination.

Cold stored PBS ( ~ 4C // -20C) should not accommodate general lab microbial contaminants to grow. Firstly, essential C and N sources are lacking and the potential of psychrophilic microbes being present under general lab conditions is highly unlikely. It's more likely that the contaminant got into your Soln when it was being carried or handled or during the frequent contamination checks and remained stationary. PBS is stable indefinitely, I have a100 mM stock for about 14 months at 4C and it was sterile, no precipitation.

Don't lose sleep over it. A way to prevent this from happening in the future is to make small aliquots of PBS that are enough for your general use, completely cover them in a single layer of foil and autoclave. I am in a Bacillus lab, and the likelihood of spores being present on surfaces is quite high. Thus far i have had no contamination issues even with my nutrient-rich buffered media.Spray the foil (if you really worried about contamination, first with 10% bleach ) and then with 70% EtOH the foil and remove it before you want to use your PBS and it should be stable and sterile. Bleach and EtOH are not sporicidal w.r.t. Bacillus but for general microbes, it should be okay.

Try it out and let RG comments know ?

Hello,

If you want to grow them in rich media, YPD or synthetic wort media work fine. If you are after some physiological characterization in bioreactors where you would like to do carbon balances or other metabolic analysis, then you might go for a more defined media like YNB with maltose or glucose as carbon sources.

With regards,

Rosa

I agree with Debapriya Sarkar in this question but you can also use some commercially available kits, e.g., FastPrep DNA isolation kit for bacteria

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

REFERENCE: https://academic.oup.com/femsle/article/273/1/35/478048

Try to read this too:

For both, in vitro & in vivo study, I have dissolved carvacrol in DMSO.

Following

Hey, I am also trying Flagellin induction, but am unsuccessful till now.. Just to ask, did it work for you? If yes, then how?

Hello, really electrical current is an important parameter to disrupt biofilm, if you use electrophoresis DC voltage 100V with about 250mA current it will definitely disrupt biofilm but also remember about superoxide radical (toxic oxygen), which can kill your microorganisms. Unter DC voltage you will have toxic oxygen which can be decreased by superoxide dismutase or catalase.

Hello guys! I am looking for the same!! Could anyone help me with that?

I agree with Alex Ignatov above “In theory, it might happen after gain/loss of T3SS in bacterium with T4SS (or v.v.) - so, it should be a rare event most probable for rapidly evolving group of strains.” Also, I am wondering about the different signals used for secretion of effectors in T3S and T4S and how they evolved.

Dear all

Genetically, genes are named based on their products/functions. Therefore, there is no gene with the name of 16s rDNA. Also, 16s rRNA sequencing doesn't indicate if the RNA or DNA is used as template. Therefore, 16s rRNA sequencing can be performed based on DNA or RNA templates. Then, you should also check the downstream technique (e.g. genomics/transcriptomics).

I think some authors use the term of 16s rDNA to make clear the ambiguity by mentioning that they have used DNA as template.

Genome extraction ----> 16s rRNA gene amplification/seq. (16s rDNA)

RNA extraction -----> 16s rRNA RNA amplification/seq. (i.e. for gene expression profiling)

Best

See attached

Negative charge and hydrophobic

We are taking up a work on milk from peri-urban areas of Guwahati city. Although there are some literature on detection of M. bovisin milk samples, I am interested on application of molecular based techniques as well as other rapid methods. How far is it possible to detect M. bovis in milk by PCR? Other than PCR, is there any sensitive and specific rapid test for detection of M. bovisin milk samples?I shall be very happy to have the answers for food safety. 

Sanchari Kundu Thank you Mam. i will look into it.,

Hi Ashwin Ramesh ,

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

I would proceed according to the protocol. The phosphate buffer is given in excess to make sure that all the calcium is precipitated. The reaction would not be at 100% efficiency if it was conducted at stoichiometric ratio. Solubility of potassium phosphates is relatively high (150g/100ml for K2HPO4 at 20C). I suggest heating the buffer solution to dissolve it completely.

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

Theoretical yield of lactic acid for homofermentative organisms is 100% (1g lactic acid per g glucose). That is 2 moles lactic acid per mole of glucose. For xylose, it is 1.67 moles of lactic acid per mole of xylose.

BUT, in the presence of carbonates used in neutralisation during fermentation, these yields can be up to 120% (>100%), due to the utilization of the carbonates as extra substrate.

Chronologically-

1) Alireza Mordadi - No quorum sensing is not a neither a signaling mechanism nor it regulates the deterministic factor for cells size determination or trigger for cell division. It is the mechanism which overall related with the population density in a particular environment. Basically it is radar homolog in bacteria. It might indirectly involve in the aforementioned, but not directly. Therefore, recommending quorum sensing (a wider topic) for a specific question is not supported.

2) Jay Sperry - since the discussion was not related to the biofilm but a bacterial growth in normal growth protocols, so I tried to relate the your previous comment to the context. And again it does not fit well, as you mentioned only one bacteria without mentioning any biofilm formation. "E. Faecalis forms biofilm after 14-15 days of incubation"(PMID: 27095620). So, what happens meanwhile, do cells divide or they remain in lag phase? And if we talk about E. faecalis biofilms, "SEM showed E. faecalis growth at all times" (PMID: 27095620).

Nevertheless, biofilms are special microbial structures, where the conventional growth curve does not applies. Therefore, involving biofilms in this discussion is like comparing apples and pears.

* I agree with your comment that bacteria might have different physiological growth phases, and it has been considered previously in the discussion above. Each species has its own growth rate and ofcourse cells requires time, a certain cell size and mass to initiate the division. But should we call it lag phase?

Hi Yaun,

Not necessarily. You design primers to amplify fragments constituting missing part of the gene. The hooks of 50 bp each are for anchoring it to the rest of the DNA. In the simplest case,

hook left- fragment left- fragment right-hook right

Length of the hook is determined by the recombination system. Without RED, you need 1 kb on each side of the gene (hook length) plus marker (kan/amp) to select for the right clone. With RED, you need 50 bp on each side of the gene and marker.

For in vitro systems, you will need linear DNA designed as before with the selection marker and typically a exonuclease inhibitor. You can do it by electroporation without RED using 1 kb hooks.

best luck,

Wes

I have utilized CTAB with great success in extracting fungal DNA from leaf litter at a very low cost. The MOBIO powersoil kit has also worked very well with leaf litter.

Are you doing direct detection from samples? If not, it's as simple as ordinary detection using primer based detection, rt master mix, no probe, non template control, and your positive control or standards, etc. You can also quantify using your standards to comparatively determine the quantity of the genes in your samples. This would be done automatically by your thermocycler, but you have to determine and insert the copy number calculated using the avocadro constant for your standards. Qualifications are only reasonable for sample based detection and less irrelevant for isolate based.

Second update: I heard back from the lab that they figured out what may have been the problem. The BamHI HF batch we used apparently may have been the problem as he finally sat down to do some more basic troubleshooting. I would be surprised if it was a HF vs nonHF problem and not a batch problem of the HF enzyme, but they ordered a new nonHF version and it worked fine. I think that was one of the only steps we didn't try before moving to infusion cloning since it was clearly cutting.

At this time, the classical approach in infectious disease diagnostics is still culture based for the majority of indications. For some indications, molecular assays are available and these are being increasingly used in the laboratories. Many companies already offer multiplex panels for a syndromic diagnostic testing approach. At this year’s ECCMID conference, we have seen a lot of cartridge multiplex PCR or nucleic acid amplification formats, enabling to test for diarrhea pathogens, respiratory pathogens, etc. Saying this, my first answer is yes, the future in infectious disease diagnostics is molecular microbiology.

However, nucleic acid amplification techniques, most likely even in POC settings, will serve as first line diagnostics, followed by culture were necessary. I therefore don’t expect a complete replacement of clinical microbiology by clinical molecular biology. However, certainly the clinical microbiology lab will undergo dramatic changes in view of the shift to molecular techniques on one side and to automation systems for culture (i.e. WASPLab, Kiestra TLA) on the other side.

I will recommend to read the latest article detailed below:

doi:10.1016/j.aquaculture.2018.02.039

Bioassay for spore polar tube extrusion of shrimp Enterocytozoon

hepatopenaei (EHP)

Hi Mustafa,

First, you need to select the disease based on the sample availability. And then the second step will be to identify the gene targets for RNA experiments, this can be done in silico using the publicly available gene expression datasets (however this requires bioinformatic skills). Other way to narrow down to the targets may be to find out some good publication (literature survey) which have identified novel target genes for the particular disease but the experimental validation is yet to be done on human samples and use these target genes for your RNA experiments after acknowledging that particular article.

Hope this helps.

Dear Milena

1. How many cells must be plated to obtain an optimal stimulation?

2. What parameter do you use for normalize Nitrite results?

3. With is the solvent for LPS and Do you sonicate LPS?

4. Do you see some morphological changes in activated raw cells?

5. During LPS treatment do you use FBS?

6. Do the cell passages influence LPS stimulation?

Good luck

Kaja

Hi Iffah,

Here are the answers for your questions, based on my experience working in the lab:

1. Close the box properly, make sure no water can go inside the box then autoclave it. You can also use tape to make sure it is sealed properly.

2. Yes, that's what we usually do.

3. Wrap them with aluminium foil, autoclave it, take them out and let it sit in the oven until it is dried.

4. Yes.

Although the microcentrifuge and PCR tubes are DNase and RNAse free, they are not sterilise. We usually autoclave all these items especially if you are working with biological stuff that can easily contaminated.

Hope it helps.

I discuss about other things. EDF is a quorum sensing signals that starting programmed cell death in bacteria and it isn't extra cell toxin. EDF in some way caused unstable toxin-antitoxin complex in bacterial cell.   

organic extraction can be tricky. try PureLink microbiome kit- it has protocols for swabs and other 

This is a late answer, but I have had success using 10 cycles. I have seen as low as 8 cycles. I have heard there are new Illumina primer barcodes that can be used in 1 step instead of 2 step PCR, which would save a lot of time and money. However, I haven't found them yet.

It may be helpful to read the book at the link.

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

There are GFP variants that have been made unstable by adding a short peptide tag that targets them for degradation so they don't hang around in the cell for very long. I've never worked with them personally, and maybe someone's improved on it since this paper, but it's probably a good place to start. It also seems like the easiest option for modifying existing constructs. 

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.

The role of the DNase is to reduce viscosity, otherwise your lysate is highly viscous and things get trapped. Sanitation or some other treatment that disrupts the DNA can be used instead of DNase, or if you are doing a gentle detergent lysis then the addition of DNase just makes the subsequent steps easier. It should not have any effect on the bacteriocin concentration but might make it easier to purify. 

By the way, many bacteriocins are extracellular and you can purify them without lysing the bacterial cells. You might want to determine whether that is the case with yours. 

Thank you Umar.

Thanks for your help - I definitely need to do more research

Using a fresh culture, you could try resuspending a few colonies in 150 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA), boiling at 98ºC during 10 min in the thermocycler and using the supernatant as DNA template (5 µl) for amplification.  It works well with Bacillus anthracis.

Using a fresh culture, you could try resuspending a few colonies in 150 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA), boiling at 98ºC during 10 min in the thermocycler and using the supernatant as DNA template (5 µl) for amplification.  It works well with Bacillus anthracis.

I have been working long time with Resazurin:  it clearly show anaerobic conditions  only when there is total absence of oxygen. Also the pH is important for Resaruin to become clolorless. The point is that flushing with nitrogen does not mean having total absence of oxygen. When I worked with liquid media, the addition of sodium sulphide caused the riduction of all the oxygen and the medium became colorless. Working with anaerobic jars, you have to add some small bags: the compound inside the bag reacting with added water, produces hydrogen, that by a catalizer bag  located in the jar, allowes the total reduction of oygen. In these conditions, the small strips of resazurin indicator become colorless. The nitrogen alone, even at high purity, contains always minor amounts of oxygen: the resazurin never will turn colorless if you do not add a compound able to transform the residual oxygen in H20. So if you need "strict" anaerobic conditions, you must add a chemical that "destroys" all the residual oxygen present.

See these papers:

Tandoi V., Di Stefano T., Bowser P.,Gosset J.M. And Zinder S.H. (1994): Reductive dehalogenation of chlorinated Ethenes and halogenated ethanes by a high rate anaerobic enrichment culture. Environmental Science and Technology, 28, 973-979.

Gatell X.M., Tandoi V., Gossett J.M. and Zinder S. H. (1995): Characterization of an H2 utilizing enrichment culture that reductively dechlorinates tetrachloroethene to vinyl chloride and ethene in the absence of methanogenesis and acetogenesis. Applied and Environmental Microbiology, 61, 3928-3933.

Dear Fahimeh,

When using the standard curve method, the quantity of each experimental sample is first determined using a standard curve, and is then expressed relative to a calibrator sample.

In order to use this quantification method, prepare five (5) 2-fold, 5-fold, or 10-fold serial dilutions of cDNA template known to express the gene of interest in high abundance. Use each serial dilution in separate real-time reactions, and determine their threshold cycle values.

In a base-10 semi-logarithmic graph, plot the threshold cycle versus the dilution factor and fit the data to a straight line. Confirm that the correlation coefficient (R2) for the line is 0.99 or greater.

This plot is then used as a standard or calibration curve for extrapolating relative expression level information for the same gene of interest in unknown experimental samples. The relative quantification calibration curve result for the gene of interest is normalized to that of a housekeeping gene in the same sample, and then the normalized numbers are compared between samples to get a fold change in expression.

A standard or calibration curve must be generated separately for each gene of interest and each housekeeping gene.

Let me know why do you want to compare the number of bacteria. As per the information given by you, it is a redundant exercise. 

At least 3 soils samples (or replicates) per plant, but it is better if you have more.

the best is to use small glass capillaries as container for frozen isolates (20% glycerol or DMSO).

It depends how you obtain fungal colonies. Do you start from one spore or just a "loopful" of mycelium? in this second case the colony diameter or area is not indicative of growth. Thus I would suggest to measure only the decoloration halo diameter excluding the diameter of the colony

From the position of the small peak close to the 5S, I would say that you are detecting the tRNAs, so nothing to worry about. You can run the sample on a PAA gel next to an appropriate ladder for confirmation, the bands should migrate between 70-90bp. 

I would rather be concerned about the great imbalance of extracted 5S and 16/23S. Is the kit/protocol you used designed for specific isolation of small RNA?

I hope that following publication will be helpful

Hi

The resistant bacteria against lactic acid Bactria different among spices of bacteria. 

example five strains of   L. monocytogenes  some strains inhibition and no inhibition either strains . The resistant bacteria  depends on several things

Do it on a microwave oven.

Also, if your autoclave is an old model (where you can remove items from the sterilization chamber while the temperature is still relatively high) you might try autoclaving directly, without melting the agar beforehand, and then homogenizing the resulting solution afterwards.

Disc or well diffusion tests are both qualitative assays. Hence, they wont give an accurate estimation of the effect of the antimicrobial activity of plants extracts or any other antimicrobial agent. Minimal Inhibitory Concentration (MIC) and IC50 values must be investigated for quantitative estimation. It all depends on what you are testing e.g. crude extract, protein extract, pure compounds or mixture of phenol-like compounds...doing well diffusion or disc diffusion test of crude or non-purified compound is meaningless... any plant extract would have some inhibitory activity due to the phenolic substances...  

Dear

My contribution

Lactic acid bacteria (LAB) are catalase-oxidase negative in general. MRS is usually used to cultivate LAB. In case you got other microorganism on your plate, it is highly suspected that you have contamination and in most cases this contamination is due to either Bacillus spp or other yeasts which can grow also on MRS. You may consider using M17 supplemented with 5% lactose or glucose. Do not use general media ! LAB needs enriched media to grow. In addition, most of G-negative bacteria can not grow on MRS. If you are not happy with MRS and M17, you can use Tryptic Soy broth supplemented with 2% yeast extract.

Keep in mind that it is not as easy as it looks to isolated pure LAB from honey or any other fermented sources.

-Abdelahhad

Stephanie, I think it largely depends on how clean (i.e. particle free) your lab environment is and what else is happening near and around you: Mold growing in the aircon and in/on fridges, colleagues happily eating crunchy food next to you and mice running around your bench (or your labemate's)?  In that case, (and in general, actually) better escape under a laminar flow, as the experiments are pretty lengthy and laborious, and the phage libraries are pretty precious and expensive resources.

There is no need to panic about biosafety however, it's rather about protecting your samples and ensuring the quality of your data.

Before you are going into the "hot phase", the manual probably suggests to get used to the techniques involved and to do some phage amplification and retrieval and, possibly, playing around with a control phage panning experiment until you are sure you want to do the actual screening. So you actually may check safely if you don't get nasty contaminations.

I take it you are sending the samples to a commercial facility with fixed standards for acceptable material? We sequence some extremely low biomass samples that come from extreme environments--some work, some don't.  If you take the risk that they won't work, would your facility make the attempt? 

Someone may suggest using whole genome amplification, but I don't advise it. Your results almost certainly will be biased. 

Hi Samuel, unless your lab uses Nile red frequently to assess lipid accumulation in multi-stain assays, I suggest avoiding it. Nile red exhibits solvatochromism in a hydrophobic environment (such as neutral lipid), and this causes a 40nm emissions to lower (more blue/green) wavelengths. As a result, it overlaps heavily with green fluorophores. I've worked a lot with Nile red (but not with algae) and I know it will be detected in FL1 of your cytometer. Depending on how much lipid (hence staining) is present, you may or may not be able to compensate the signal from Nile red out of the SYTO signal. If you have the money for it, you can use other BODIPY or LipidTOX dyes that emit in the red and stain lipid.

In terms of actually doing the experiment, Arno has good suggestions. To check for spectral overlap of the fluors, you can use samples stained individually with each fluor to set the compensation parameters (your flow technician should know how to do this).

When you process your data, I suggest using FlowJo. You need to buy it , but its very good software. I havent used them, but free softwares are Flowing and CyFlogic.

Hope this helps!

Here's a paper for you on Nile red:

Hey Katya!

I would recommend using the "bbmap" tool included in the "BBTools" suite for that task.

For an introduction to the "bbmap" tool: 

For a description of how to use bbmap to remove human contaminant DNA (also includes an already processed reference genome available for download):

Make a heavy suspension of your culture in glycerol broth (20% glycerol) and place the vial as soon as possible at -80°C. When needed scrape a little material from the surface of the froozen stock (prevend thawing of the stock) and culture in a suitable growth medium. Your strain will stay viable for many years this way. Using small srewcap vessels with one to one and a half milliliters glycerol broth will be enough.

To evaluate the antibiofilm efficacy of different matherial such as titanium surfaces, the whole biomass present on each disc was detected. Briefly, biofilms grown on titanium

discs were air-dried and stained by disc immersion in a 5% crystal violet solution for 15 minute and, after several washing , air dried again. The estimation of biofilm biomass was performed by elution of the biofilm bound crystal violet with ethanol (96%)

followed by the determination of the absorbance of 100 ll of eluted dye solution at 595 nm using a microplate photometer (Multiskan FC, Thermo Scientific; Milan, Italy). Measurements for each discs, were carried out in triplicate. For each bacteria and

surface, mean values and standard deviation of absorbance value were computed. Un-paired Student’s T test was used to compare data between experimental surfaces.

I hope this protocol help you

I do not know how far it be relied upon the supply of urease as mention by Mozafari. If I were you I would go in for isolation of the enzyme form the culture of H.pylori which would definitely very reliable for my research work. The isolation of the enzyme is pretty easy.

First, I think it's good result for negative control. The Xv products are really good, but your problem is the false positive to other DNA, right? So, my comment is may be you can decreased the incubation time to be 30- 40 min because from you picture it's seem like the Xv product is very strong while the others are weak or low concentrations (except Ea and Cms). or may be you can increase temp. to be 66-67 C. 

PS. I have question about the concentration of the outer and inner primers in the reaction why you use the same concentrations (1.6 uM) or it is your mistake of typing information. Because normally the inner primers should be more than outer primers 8-10 fold. Good luck for you.

Hi Krishna Kumar Sharma, I worked with a few antimicrobial peptides (Their role in immune modulation). Sometime during my study I planned to visualize the peptides in the cell membrane (or in cytoplasm), but dropped it because my goal is to see the immunological status of the mammalian macropage in the presence of the peptide. If you are trying to see the peptide aggregation on bacterial surfaces through florescence microscopy, label the peptide with Cy3 (Red) using protein labeling kit  (GE health care) and bacteria with CFDA/SE (Invitrogen) (Green) vital dye. Incubate the labeled bacteria and peptide together for 1 hour. Fix it with 2% para-formaldehyde and mount it on glass slide to see it under Confocal or florescence microscope. You can see the bacterial labeling with CFDA/SE here https://www.researchgate.net/publication/299576966_Comparative_Analysis_of_the_Effects_of_Two_Probiotic_Bacterial_Strains_on_Metabolism_and_Innate_Immunity_in_the_RAW_2647_Murine_Macrophage_Cell_Line. All the best for your research.

Hi Erica,

Could you include a picture of your gel to give us an idea how close together the bands are? Following on from Julian and Ali's questions, you need to work out if the multiple bands are

DGGE is a massive pain to work with. If there is any way you can change that part of your experimental design, I would highly recommend doing so.

Also, one common problem with community analysis is that your answer is very dependent on the primer set you use. It's worth checking that the set you have selected is adequate at amplifying your target fungi so you don't unintentionally loose some information. Depending on what taxon resolution you're after, ITS may be too short a sequence to give you an ID down to species level when you get to the sequencing stage.

All the best!

Hi Alex,

Thank you again for clarifying things.

I will inquire about the database before starting to identify my bacterial strains.

Have a good day,

Meriem

Hi Jorge

Thank you so much for your help. Just a few more things, should i do an OD before i plate them or just plate them after 24hrs ? Also should I test the them on selective agar just to make sure the bacteria is the strain i want ?

Thank you for your response, I have used the aforementioned Difco marine agar and those plates appear to stimulating better growth, I have also noticed an increase in colonies by changing the incubation temperature to 28 degrees.

Sim Huey Yi:

The following reference may help you.  The full article should be available through open access.

Girardin H, Latge JP, Srikantha T, Morrow B, Soll DR. Development of DNA probes for fingerprinting Aspergillus fumigatus. J Clin Microbiol. 1993;31:1547–1554.

Thank you @Ali Mahmoudpour for sharing your experience. 

Here's a PDF of the original paper describing the construction of pRK2013, not as nice as having the actual sequence but hopefully it helps. 

(If I'm reading this right, digesting with PstI and HindIII should produce a fragment ~7.2 kb.)

Hi Caroline,

I co-cultured Raw264.7, THP-1 and HCT-116 cell lines with Lactobacillus acidophilus, Bacillus clausii, Bifidobacterium bifidum and Saccharomyceses cereviseae to find out their role in activating the host innate immunity and with Salmonella typhimurium to find out its cytotoxicity and inflammatory property on macrophage. You can find the paper here (http://link.springer.com/article/10.1007/s12602-016-9211-4).

You need to use a solution with very high concentration of guanidinium thiocyanate or guanidinium HCl, as Silica beads or columns (filters) are pretreated with strong acids to activate that capture of nucleis acids, and later on is important to keep in washing solutions very high salt and alcohol concentrations to avoid release. Some hints, working solutions and trials at:  https://arbol.uniandes.edu.co/Archivos/Ivanova-Membrane-Based%20Protocol.pdf