Silvia_84

Hi all, I got nucleotide sequences of a PCR product corresponding to a specific variant of an enzyme I am studying. I made a search of homologies by BLAST to see if the gene I have shows 100% homologies with already known variants or if it is a new variant. I found some changes in the sequence and I would like to know if this change is just a silent mutation or it leads to a different amino acid sequence. How can I check this? Do you know good softwares for the translation of the DNA sequence in amino-acids? Thanks

Hi all, I am new in Microbiology lab and I was reading about the conjugation assay that is done to see if the resistance is transferred among bacteria. I was wonder..can this test work if the plasmids are not transmissible? How reliable is this test in testing if the plasmids are transferred?Do you have an idea? Any suggestion is appreciated. Thanks

Hi all, could you explain to me why the carbapenems (and other antibiotics) are considered last-resort drug? I am aware that they are used when all the other drugs fail to treat infections but I don't understand why they are the strongest..Do they have more catalytic activity?Do they act with particular molecular mechanisms?Can you explain me please? Any suggestion would be really appreciated. Thanks

Hi all, can anyone suggest me a method-protocol to understand if a gene is on the genomic or plasmidic DNA?I have done PCR on purified DNA (that is both genomic and plasmdic)and I found the gene I was looking for. Now I would like to understand if this gene is located on the plasmid or on the genome. How can I do that? Thank you

Hi all, I tried to do multiplex PCR to amplify more genes at once with the biomix red polymerase. Unfortunately it did not work (no bands at all, not even primer dimers)and I have no idea of what goes wrong. The primers should be ok because I am using those already used by another group who has already done the same multiplex PCR.I don't ' know if I make some mistake in the procedure: For a 25 ul of reaction mixture I added: 12.5 ul of BIOMIX RED 1 ul of each primer (they are in total 8 because the genes I am amplifying 4 genes together) 3.5 ul of water The single PCR, done with the primers separately is fine.. so there must be something wrong in the multiplex set-up Thank you in advance.

Hi all, I have some stocks of bacteria frozen with beads. I was wondering what method is used to streak the beads on the agar plate.Do I need to shake a bit the plate to let the beads rub on the agar?Or are you aware of other more accurate techniques to do it? Thank you in advance.

Hi guys, I have two questions about the preparation of bacterial freezer stock with beads.It is new for me. How can I make it?Can you indicate me the procedure/ steps to carry it out? When I have to streak the bacteria from the frozen beads , how is it better to do it? roll the beads on an agar plate or just use a loop to touch the beads and pass it on the plate? Thank you very much.

Hi all, I am doing PCR and I am using the biomix red as a polymerase. The mix already contains all the components for the reaction but I need to increase the concentration of magnesium in my reaction. I can't understand very well the protocol. It says that the MgCl2 concentration in the buffer provided is 6mM (3mM final concentration) and should be adjusted just if necessary. Then it reports a table showing the volume of additional MgCl2 to add to a 50 ul reaction to achieve different final concentrations. The table says to add 0 ul of 50 mM MgCl2 to 50 ul final reaction volume to reach a concentration of 2mM, 0.5 to reach a concentration of 2.5 and 1 for a 3mM concentration. What I don't understand is that reading this protocol it seems to me that the reaction mix already contains 3mM of Magnesium. So why should I add 1 ul to reach a final concentration of 3mM?And if I wanted a 5mM concentration, should I add 3ul? I was not able to attach the link, anyway if you google biomix red protocol , you find it right now. I hope to have been clear. Thank you

Hi guys, can I ask what I should do if I want to order some bacterial control strains for PCR? What is the procedure? Do I need the genomic sequence of these strains?Are there websites for this? Thank you very much

Hi all, Do you have experience in multiplex PCR? Do I need a specific polymerase for this kind of reaction? Thank you,

Hi all, I extracted DNA and now I have a stock solution in TE buffer and two aliquots in water. How is it better to store all these solutions since I need to use it for long periods of time? Freezer or fridge? Thank you

Hi all, I have to do PFGE on some strains of Klebsiella. It is the first time I do it and I am reading a protocol for the prepararation of agarose embedded bacterial DNA. The procedure says at a certain point to remove 5 X 108 cells (I suppose they refer to a bacterial suspension) for each ml of agarose plugs to be made. Can you explain me how you determine the volume of bacterial suspension corresponding to this amount of cells (5 X 108) and how is it calculated? I need to know it even for other protocols that say to take a specific number of cells and I did not understand how to convert this amount in volume. I thank you very much.

Hi all, I have to run a PCR reaction and I am following a protocol suggested by the company of the TAQ polymerase I have. The protocol says to use 10 ul of 5x MyTaq Reaction Buffer and 0.25 - 1 ul of 2XTAQ polymerase. in the reaction Does it mean that I have to diluite both before using them since they are concentrated respectively 5X and 2X? Or shall I take the amounts suggested by the protocol from the concentrated solutions as provided by the company? It is not specified on the protocol.. Furthermore, I did not understand very well how I can calculate the volume if TAQ polymerase I need if the concentration is reported in Units.. e.g. If the protocol says to add 1.25 units of TAQ, at what volume does it correspond?Can you explain me a little bit the calculations I should do? Thank you very much in advance

Hi all, I was wondering if you can illuminate me or give me some opinions on what can go wrong during my PCR reactions. I extracted bacterial DNA and I diluited my DNA samples 1:10 and 1:100. I tried to run different PCRs to identify a gene. I run a PCR by using the extracted DNA as it is, another one with the DNA diluited 1: 10 and another one with DNA diluited 1:100. I was able to see the product if the DNA was dilutied 1:10 and 1: 100, but not if I used the extracted DNA as it is. I thought that maybe something with DNA extracted during the PCR reaction can go wrong because the DNA is too concentrated, since in the other two cases (when DNA is diluited) it works ...does it make sense for you?Or do you have other suggestions? I tried to run again these same PCRs, same conditions but this time I am able to see the product with the DNA extracted and when it is diluited 1:100 but not if it is diluited 1:10...Do you suggest something?What would it mean? Thank you in advance

Hi all, Do you usually centrifuge your samples containing the mix for the PCR before running it? Thank you in advance

Yes, sorry. I was just asking what type of dilution is better in terms of quantity.. if to do 1:10 in water or other..sorry I am new with this stuff.

Hi all, I should measure the DNA concentration I obtained after the DNA purification. I have a total volume of DNA solution of 100 ul and I would like to use the spectrophotometer. Since I have the 10mm cuvettes, how can I handle the volume of DNA to test in the cuvette? I mean, the reading measures will be done on 1 ml of solution into the cuvette, won't they? Should I dilute my DNA sample in water in order to fit the solution in the cuvette and measure the concentration ? If yes, how do I diluite it? Thank you in advance.

Yes, sorry , I did not explain appropriately. The bands are so thick that they overlap and the separation is not visible..the ladder appears as a continue vertical line in certain points. I prepared a 1.5 % of agarose gel in 1XTAE buffer.

Hi all, im am new in doing electrophoresis on agarose gel. I tried to run a couple of gels and my markers are not separated appropriately. I have to say that the bands corresponding to both the marker and to my DNA samples are so large that I am not able to interpret the results. Even if I run the gel for more time, I obtain always the same result. On what it could depend? on the amount of sample I load? I usually load 5 ul of sample. Thank you in advance.

Hi all, I have a very stupid question.Is it important the side of the combs when you place them in the tray for the agarose gel to form the wells before pouring the gel into the tray? Can you explain me why? Thank you so much.

Hi guys, I am following a protocol for the DNA extraction from Gram negative bacteria. I am completely new in this stuff.The protocol says to start from a bacterial suspension of approximately 109 CFUml . My question is : How can I determine and check if I have this concentration?with the spectrophotometer? if yes, how can I get the concentrationj from the absorbance? Thank you very much.

Hi all, I have a few questions about the purification of DNA. I followed a protocol specified in a kit for the DNA extraction and purification. The last step of the procedure says to rehydrate the DNA in a rehydrating solution (already present in the kit). I have done it but I have difficulty to dissolve the DNA and make it go in solution. I am able to see the DNA pellet. My supervisor suggested me to flick the tubes just with my finger to try to dissolve the DNA..but the pellet is still not dissolved.Can you suggest me something to get the DNA in solution? My second question is: how can I determine if the concentration of DNA (after purification)is good and significant (in terms of amount)for next experiments? Are there some range of values of concentration to consider as good for the DNA? Is it better to diluite the DNA before using them? Which dilution? 1:10? Thank you very much in advance. Silvia

Hi all, I have to prepare a stock solution of ciprofloxacin that is soluble in dilute aqueous acid. I did not understand what dilute aqueous acid is and how I prepare it. Can you give me some suggestions? Thank you in advance. Silvia

Hi all, I have a question about a calculation I am struggling to solve. I have two mix two different antibiotic solutions..one at 50mg/ml and the other one at 10mg/ml . The final solution of the mix of the two antibiotics should be at 10 mg/ml...How do I calculate the initial volumes to take from the two stock solutions to mix? Thank you in advance. Silvia

Hi all, have you ever tried to re-run an agarose gel after the run is finished and after visualizing it under UV? Last time I run a gel for a hour but the bands were not separate properly yet. I made the gel again but maybe (for a next time)it is possible to put the same gel on a second run after the analysis? Or does it get degraded?If yes, can you also explain me why? Thank you very much. Silvia