Biochemistry and Molecular Biology

Hi all, I would like to ask if you know about molecular technique to quantificate intracellular bacteria from biopsy samples. Flow cytometry require labing of bacteria, which is not possible if you are testing biopsies. Another option which popped into my mind is qPCR - however how would you overcome with possible DNA fragments in sample? Is there any other technique you would recommend? I will by really thankful for any help.

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

Hello, I'd like to extract an arthropod from a biopsy of animal skin for identification. The mite is small, about 100um, and embedded in a column of tissue. It's too small to manually cut out under magnification using available tools. It's currently preserved in 70% ethanol. Ideally I would isolate it by loosing or dissolving the surrounding tissue without destroying its features. I'd like to conduct a visual inspection prior to sequencing for species identification. Specific suggestions and techniques welcome. Thanks. See photo: http://s32.postimg.org/4nm6krc79/specimen_2c.jpg

Hi I have a problem in the expression of plant viral gene in E. coli system. The gene is cloned in pET28a and the sequence is in frame (confirmed by sequencing). I have tried the expreesion in DE3 (BL21), PlysS, Rosette PlysS, but could not get the expression. The expected sixe of protein is 48 KDa and 14 KDa. Kindly suggest me some troubleshoot.

I doing experimentation for peptide prediction using machine learning. I need some data for testing. I already have one dataset, but I need another one or two for verification. I'm looking for one a data for proteotypic peptide and non-proteotypic peptide.

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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 am conducting a group research project currently and I am seeking the answer to how Chloro-xylenol (4-chloro-3,5-dimethylphenol) kills bacteria and extensively kills amphibious organisms. I have found numerous articles stating that this chemical does in fact kill these organisms, however does not go into detail of how. Noone seems to know at this stage. So far I have found that the chemical can disrupt an organism's membrane potential leading to a complete lack of ATP production, however this information was discovered from a questionable source. Any thoughts or comments would be much appreciated. Thanks, Andy

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 m…

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 concen…

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! I am doing an experiment for my Diploma Project where I analyze and compare a few samples of DNA (all from the same source, my saliva) that have stayed outside for various amounts of time before being frozen. I chose to let the samples stay with no lid on but I am now facing the problem of evaporation, and I have no clue how to proceed in rehydrating the pellets after they have been thawed. I have tried researching on this but found no credible/relevant sources. I am not very experienced in this subject so I might have been searching for the wrong things so if anyone could direct me to some protocol or help in any other way it would be extremely apprecia…

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

Hello, my current PhD research INVOLVES a detailed Proteomic study of a class of detoxication enzymes - GSTs. I'm running out of time to meet the deadline but I need to carry out Amino acid sequence of this protein. The feeders i'm getting on the cost is outrageous and i believe there are people in this scientific community who has probably done protein sequence before and can give me an advice on how to get a cheaper cost or maybe a fellowship.

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…

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 ..…

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

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.

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.

Hello to all! I'm a newcomer here, please keep in mind: my English is far from good, so be patient ! All suggestions appreciated! I didn't find answer searching the forum, so I'm asking here: It is about EDTA (disodium-EDTA-dihydrate salt). I need help - how to make solution of that chemical for use as human blood (peripheral whole blood) anticoagulant (in vitro), and then, how to make working solution for dilution of blood sample so I can use it in hemacytometer! I mean: to make EDTA sol. first, and then use that solution for diluting blood sample. Should I add something to EDTA sol. before blood dilution (wrong pH for example)? I can only find Na2-EDTA and "EDTA free…

Comrades who knows please tell me how an ordinary technician in the lab to get artemisinin-transferrin conjugate. I need in transferrin molecule covalently assemble iron (II) and artemisinin. Please describe the process action in order to get the final result, it is necessary for injection. The idea is to create a medication for cancer treatment. Or give a link to another forum where to me can probably help. I has artemisinin 98%. But second question is how to check whether or not a real artemisinin or other substance? And the third question, describe the process in the experiment to produce artemisinin from Artemisia annua. Thank you, I hope here is many geniuses who…

Whats an advanced textbook on Nutritional science or sports nutrition for people who already have a solid understanding of biochemistry. Recommendations much appreciated!

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 …