69 Questions 43 Answers 0 Followers
Questions related from Silvia Caprari
Hi, I plotted my data in a bar chart (I used GraphPad) showing the average values, the standard deviation error bar and the single point values. I can't understand if the points that are plotted...
06 November 2018 4,218 4 View
Hi, I am using the software PyElph to investigate the similarities between RAPD profiles of a few bacterial isolates. My question is: Which algorithm is it better to use to generate the...
02 October 2018 9,159 1 View
Hi, I have a question about the 0.5 McFarland standard. In different websites I read that the for the 0.5 McFarland standard, the absorbance at a wavelength of 625 nm should be 0.08 to 0.13. I...
21 September 2018 5,750 1 View
Hi all, i am new in image processing and using ImageJ. I want to measure the density in areas of pictures that were originally taken at a different magnification. I attached an example that I hope...
06 June 2018 4,655 3 View
Hi, I am new with Western Blot and I need a few clarifications about the use of blocking and incubation solution. I understood that blocking is done to saturate the membrane with proteins...
06 December 2017 9,132 4 View
Hi, I am preparing a sodium phosphate buffer solution at 0.1 M pH 7. For the preparation I am following this instruction that requires to mix 1 M Na2HPO4 and 1 M NaH2PO4 like this: 57.7 ml 1 M...
13 November 2017 9,488 3 View
I have a question about the calculation of the enzymatic activity from the absorbance. In particular I have a series of curves corresponding to the different concentrations of my samples. The...
03 October 2017 4,804 4 View
Hi, I have a question about how to calculate the enzyme activity from absorbance. I have absorbance values for different concentrations of my samples at different times. As far as I understood, I...
26 September 2017 7,039 3 View
Hi all, I would like to ask you a question about the data analysis in an enzymatic activity assay. It is the first time I deal with this kind of stuff. I have a kit for the beta lactamase...
25 September 2017 3,595 4 View
Hi all, I made different minipreps starting from the same sample of bacterial culture because my intention was to increase the concentration of plasmid in my final plasmid preparation. Now I have...
22 June 2017 9,021 3 View
Hi all, I have a few questions about ligation 1)Could you explain to me why you need an excess of insert compared to the vector?What is the “chemical principle” of this reaction? 2)In my...
14 June 2017 8,125 6 View
Hi all, I am new with cloning and I am trying to carry out a ligation with three elements (two inserts and the vector). All of them must be cut with restriction enzymes and run on the gel before...
06 June 2017 6,174 5 View
Hi, I am running PlasmidFinder on fasta files containing assembled contigs. The results seem to indicate the presence of two replicons in the same contig even if I am not completely sure of how...
24 May 2017 7,425 3 View
Hi all, I have set of data relative to MIC values of different isolates for a set of antibiotics. Let's say that I have the excel file in attachment where for each strain the MIC values for each...
26 April 2017 2,657 1 View
Hi, I need to make a construct for protein localization. The construct should be made of the gene of interest and the GFP gene. I don't have idea on how to stick the fragments together with the...
19 March 2017 8,905 4 View
Hi all, I was wondering.Given the DNA sequence of a protein in bacteria, how can I determine where the signal peptide is in this sequence? I found servers that predict the position of the signal...
16 March 2017 6,145 3 View
Hi , I was wondering if after overnight incubation the bacteria are still in exponential phase or in stationary phase. Thank you very much
06 March 2017 5,469 5 View
Hi all, I have a few questions about next generation sequencing and the generation of the reads. I did not understand some parts of the process..In particular, are there more reads generated for...
08 February 2017 5,782 3 View
Hi, can you explain me why the minimum inhibitory concentration is measured after overnight growth of the microorganism? Actually, I thought the MIC was measured during the log phase since the...
20 January 2017 7,633 15 View
Hi , I am new with the Illumina sequencing and I got back the sequencing data. I am quite new with the terminology. Can you explain to me what it means that a project will be sequenced using...
10 January 2017 7,701 12 View
Hi all, I extracted RNA from bacteria and I run an agarose gel on these samples. How do I recognize if I have RNA and not DNA? I have three bands (in attachment) and I am not sure about what they...
22 December 2016 8,059 13 View
Hi, I can't understand well the use of the normalization for analysing reverse transcriptase real-time pcr data and why you need to use housekeeping genes. Actually my issue is that I can't...
13 December 2016 6,410 4 View
Hi, can you explain to me (in simple words) why i need to normalize my values after a RT-qPCR? In my case, I am studying how the expression of antibiotic resistance genes changes when the cells...
07 December 2016 3,451 6 View
Hi all, do you have experience in using PyElph to analyse gels?I want to use it to analyse my RAPD gels images and build a dendrogram. The question is : I have the RAPD profiles for...
08 November 2016 5,470 0 View
Hi all, I am trying to design primers (I am using Primer3) for RT-qPCR on a bacterial gene (so the reaction will first do cDNA with reverse transcriptase and then qPCR). I am following a tutorial...
07 November 2016 3,413 1 View
Hi all, I was trying to understand if the entries with extension "complete genome" contain both the chromosome and the plasmid sequences of the bacterial strain the entry is referring to..or just...
03 November 2016 9,858 1 View
Hi all, are LB broth and Nutrient broth minimal or complex media? I am following the QIAGEN protocol for the RNA extraction from bacteria and the protocol for the preparation of lysates is...
02 November 2016 6,261 3 View
Hi all, I am completely new in the field of Western Blot. I need antibodies directed against the bacterial gene "aac(6`)-Ib-cr" that codifies for a protein that is a fluroquinolone acetylating...
31 October 2016 3,836 4 View
Hi all, I have to design primers for qRT-PCR on a bacterial gene. I am aware that one should pay attention to exons/introns..but since I am dealing with bacteria, do I need to care about this...
31 October 2016 3,495 7 View
Hi , can you explain in a very very simple way why the repeat sequences interfere with the assembly of the contigs in the sequencing? I can't figure it out. And why the pair-end sequencing helps...
19 October 2016 5,429 4 View
Hi, I am checking if my bacteria are resistant to particular antibiotics. I don't want to do the MIC, I would like to have a rapid result since I am testing several antibiotics. What I have done...
19 October 2016 8,678 5 View
Hi all, how do I choose a genome to be used as reference if i want to order the contigs once they are assembled?I know that it is supposed to be the most closely related . But, for example, in my...
04 October 2016 636 5 View
Hi all, I wanted to ask you where it is better to keep bacterial overnight cultures if you don't use them immediately. Let's say that I have overnight cultures and I want to streak them on plates...
28 September 2016 3,535 3 View
Hi all, I am digesting with a double digestion XbaI and BamHi the plasmid extract of different clinical bacterial isolates to see if they have the same plasmid profile or not. In the small gel, I...
20 September 2016 9,195 3 View
Hi,I am trying to visualize my DNA bands run on agarose gels (stained with gel red during the gel preparation). Although I am using UV fluorescence to visualize the gel, I think some setting in my...
15 September 2016 1,283 3 View
Hi all, can you tell me what tubes it is better to use for broth mating conjugation experiments? Is it better to put or not a lid on the tube? I read that the choice of the tubes can affect the...
25 August 2016 7,624 6 View
Hi, I am studying genes for the resistance to the antibiotics in Klebsiella and I am analysing an outbreak of carbapenem-resistant isolates. The gene I am analysing is OXA-48 and it is known to...
23 August 2016 942 3 View
Hi, do you know a protocol for bacterial broth mating experiments? Thanks
23 August 2016 8,593 3 View
Hi, I am analysing some data coming from genotyping I am performing on carbapenem- resistant isolates of K.pneumoniae. The resistance genes should be carried on plasmids and I looked for the...
22 August 2016 3,577 1 View
Hi, I have to do a bacterial conjugation experiment for the first time. I don't have so much idea of how to set the experiment. My donor strain is a carbapenem resistant isolate and the acceptor...
22 August 2016 9,557 4 View
Hi, I am trying to get plasmid profiles from clinical isolates. I extracted them and visualized them in the gel electrophoresis. I tried to cut them with a 6bp enzyme XbaI to have more ideas on...
19 August 2016 6,550 2 View
Hi ..I run an agarose gel on a plasmidic extract and I have different bands. I don't know if they are different forms of the same plasmid or different plasmids. I want to determine if the bands...
16 August 2016 4,720 2 View
Hi, can you tell me what a good transformation efficiency for electrocompetent cells is? Thank you
15 August 2016 7,278 1 View
Hi, I am studying the resistance to antibiotics in bacteria and I often happen to read papers where the authors report minimal inhibitory concentration tests performed on these resistant bacteria....
31 July 2016 3,702 5 View
Hi, I heard about the use of IC50 to test the susceptibility of a bacterial strain to a given drug. I didn' t understand very well why people use it to test the acceptor strain before performing...
31 July 2016 4,630 1 View
Hi all, i am trying to do transformation with plasmidic extract from antibiotic resistant Klebsiella to DH5a cells. The plasmids should carry the resistance genes, so I am trying to see if this...
31 July 2016 4,000 5 View
Hi, I have to do conjugation experiments to see if the resistance is transferred by plasmids. I don't have a strain in lab to be used as acceptor for my conjugation experiments, so I have to buy...
28 July 2016 1,612 3 View
Hi, I am considering to buy MAX Efficiency DH5 alpha Competent Cells to try to do both electroporation and heat-shock experiments with large plasmids. I don't know what procedure will be better,...
27 July 2016 9,958 6 View
Hi all, I performed electroporation with bacterial plasmidic extract for the first time to confirm that the plasmids carry resistance genes. Nevertheless, it didn't work. I had no colonies on the...
23 July 2016 3,344 12 View
Hi all, can anyone explain me why for some procedures it is better to use bacteria in log-phase and for other ones bacteria in stationary phase? For example for DNA and plasmid purification you...
20 July 2016 2,455 6 View
Hi, I am doing transformation experiments with plasmidic extract and I am using DH5a cells for the transformation..I obtained few colonies on a plate with 1 ug/ml of meropenem antibiotic. Anyway,...
19 July 2016 9,097 3 View
Hi all, I have to run a gel to separate small fragments, 50-25 bp..What are the best running gel conditions (in terms of time and voltage)? I did not understand very well how you choose the time...
12 July 2016 3,181 6 View
Hi , how long can I keep LB plates containing Ampicillin and X-gal in the fridge? Thank you very much.
12 July 2016 1,531 6 View
Hi all, I run a PCR to look for an integron in different bacterial isolates to test the presence of this integron in the bacterial genome. This integron is often associated with resistance...
11 July 2016 4,333 3 View
Hi alI, I have a general question about the molecular cloning. I have to clone the genes for the resistance to the antibiotics in E.coli and select the transformant clones. I was wondering if for...
01 July 2016 980 4 View
Hi, I have to prepare a solution reported like this in the protocol I am reading: 0.1 M Maleic acid, 0.15 M NaCl; pH 7.5 (20° C); 0.3% (v/v) Tween 20 How do I interpret these instructions in your...
23 June 2016 6,771 2 View
Hi, I need to generate DNA probes for Southern blotting experiments. In particular, what I am interested in is looking for a specific gene in plasmids of a set of bacteria. So, my probe should...
23 June 2016 3,237 3 View
Hi all, I am struggling with exctraction of plasmids from some isolates of Klebsiella pneumoniae. I am using a manual extraction protocol that I am attaching in this message. Last week this...
21 June 2016 8,391 3 View
Hi all, I am extracting natural plasmids from my bacteria. I don't know the size, nor the sequence. I am trying to determine plasmid profiles of the bacterial isolates I have. I have bands on...
20 June 2016 2,818 5 View
Hi all, I am trying to extract natural plasmids from my bacterial strains. I don't know the size of plasmids, nor if different plasmids are present in the same strain. I run a gel with the...
14 June 2016 6,137 7 View
Hi all, in your opinion is it a problem that I have an EDTA solution at 9.5 instead of 8 for plasmidic extraction? I added more NaOH to dissolve the powder because at Ph 8 there still were not...
08 June 2016 6,713 2 View
Hi all, I needed to prepare an alkaline SDS solution made of 1% SDS in 0.2 M NaOH. What I did is to dissolve 1 g of SDS in 100 ml of 0.2 M of NaOH. Is it correct? What does 1mg/ml of lysozyme in...
08 June 2016 5,350 3 View
Hi all, I have to separate bacterial plasmids (after extraction) on agarose gel. Which electrophoresis conditions would you suggest me to use (in terms of hours, voltage)? Thanks
03 June 2016 6,249 5 View
Hi, I need to calculate the appropriate hybridization temperature for my probe (DNA probe). The equation used to calculate it requires the knowledge of the % G+C and the length of the hybrid in...
31 May 2016 6,514 3 View
Hi, can you describe me how an hybridization oven works for southern blotting and where/how I put the membrane in it? I need to do a southern but it is the first time and I am not supervised , so...
31 May 2016 6,865 4 View
Hi all, I need to add 10 ng of DNA in a reaction vial and add water for a final volume of 15 ul. I start from my DNA extract which is at a concentration of 937 ng/ul. So if I want to calculate the...
27 May 2016 5,631 3 View
Hi all, can anyone suggest me a method-protocol to understand if a bacterial gene is on the genomic or plasmidic DNA?I have done PCR on purified DNA sample (that contains both genomic and plasmdic...
25 May 2016 7,528 4 View
Hi, I am studying the antibiotic resistance and I am amplifying a class of integrase genes that are known to be associated to multi-drug resistant phenotypes.I have done PCR with primers for this...
25 May 2016 1,319 5 View
Hi all, I anticipate that I am completely new and for the first time I have to do cloning of genes whose sequence is unknown (The final objective is indeed to sequence the gene). Since I am not...
23 May 2016 7,160 1 View
