Biochemistry and Molecular Biology

There is a total of 8 wells and the the two ends of the wells are not used, meaning that only 6 wells are used in this experiment. The first four wells are filled with markers while the next two wells are are filled with DNA samples. Below are the amount of markers and DNA which are filled in their respective wells (in microL): M1 : 1 M2 : 3 M3 : 5 M4 : 10 DNA 1 : 3 DNA 2 : 5 This procedure is carried out to measure the concentration of DNA. Now, what I don't understand: 1. what is the function of the markers 2. why are four markers needed 3. how do you know how many microlitre of markers needed in each well (why 1, 3, 5 and 10) 4. why do you need two DNA samples when t…

Hi everyone Just to get to the point: Hematoxylin is a dark blue or violet stain that is Basic / Positive. It binds to BASOPHILIC substances (such DNA/RNA -- which are Acidic / Negative).DNA/RNA in the nucleus, and RNA in ribosomes in the rough endoplasmic reticulum are both acidic because the nucleic acid building blocks that come off the phosphate backbone are negatively charged. These form salts with basic dyes containing positive charges. Therefore, dyes like haemotoxylin will bind to them and stain them violet.Eosin is a red or pink stain that is Acidic / Negative. It binds to ACIDOPHILIC substances (such as proteins -- which are Basic / Positive).Most protei…

Hi all, I'm a Master's student currently working in a lab which has been using vectors encoding shRNAs for transient knockdown in C2C12 cells. We're considering creating new vectors encoding AgoshRNAs to increase knockdown efficiency/ noise reduction, but since the science behind these hairpins is fairly recent, I was looking to get some insight from anyone who's used them - are there any problems associated with their use, and do they function as well as they should? I'd appreciate any advice!

Hey all, My background is in Chemistry, but I did just recently sign up for courses to earn a masters in biotechnology. I have done some research into vaccine's and I understand the general concept. I'm looking for any third party research information on vaccinations (how they behave on a molecular level) and any info on proving that they are close to 100% effective and do not cause long term (chronic) health problems. I would also love to hear your opinions and understanding of vaccines. Thanks Sincerely, I need to know.

Hi, I've been looking everywhere for the substrate preference of SHMT enzyme but can't find it anywhere. I know the reaction serine --> glycine is reversible but need to know when the forward reaction occurs and when the reverse would occur. Thank you very much!

Hello, I have ordered primer set according to the source article, as well as several other articles, should yield a 740 bp PCR products, but when I performed the protocol according to them I get 500 bp PCR products bands when run in gel. When I searched about that I have found another article where they have gotten 600 bp PCR products using the same primer set. The articles with 740 bp: http://www.apocpcont...emant K Bid.pdf http://biomed.cas.cz...5/zajickova.pdf The article with 600 bp:http://www.lifescien...802_120_131.pdf My PCR products and ladder information are the two attached images What do you suggest I do to make sure if my PCR prod…

At the isoelectric point, protein have no net charges and therefore will be less soluble. Why does increasing ionic strength result in an increase in solubility? Do the ions form a solvation shell? Also when salting out the ionic strength of the solution is increase (salt = more ions) and this results in a precipitate. The ions disrupt the protein/solvent interaction and cause the protein to aggregate and therefore precipitate out. Can someone please help explain this concept of protein solubility with ionic strength at isoelectric point. Thanks

I was looking for a type of organism in which the was no differentiation between the gametes, both are 100% identical (of course, there could be mutations, but no allele differentiation in matching-type regions, like the positive + and negative - mathing types in some algaes), and with a sexual reproduction. In isogamous organisms we have a small genetic differentiation between the gametes, as the + and - one I said. Is there an organism like this in nature? Also, is there any isogamous organism in which the gametes are produced in the same organ or region?

I have heard people state that getting RNA from t-cells is difficult. They attribute this to the fact that as the t-cell is very small it does not contain very much RNA. Now, while I have never said anything I find this logic to be sad. There should be no difference between the total amount of RNA based on the size of a cell. Every cell of an oragnism has the same amount of DNA (with the exception of erythorcytes) so unless they are metabolically more active or more actively transcribing shouldnt their be about the same amount of RNA.I might believe an argument be made that an inactive cell has less RNA than an activated cell but the argument about the size seems t…

So I have a question about the active form of insulin. I know the 1 letter amino acid sequence for insulin is: GIVEQCCTSICSLYQLENYCNFVNQHLCGSHLVEALYLVCGERGFFYTPKT I used http://web.expasy.org/compute_pi/ to calculate the theoretical pI of insulin which is around 5.39. The question is what the charge of this insulin would be in a pH solution of 7.4. The exact question is: At pH 7.4, what would be the charge (if any) for insulin? Explain how you came to that conclusion. I know when in a pH solution that is higher than the pI value of the protein then the amino acid will be deprotinated and migrate to the positive side. So would the answer be that it is negat…

Hi new to the forum, I was helping my girlfriend out with some homework and what I am reading in her bacterial pathogenesis book is a bit different from what I learned. Basically, I was wondering how you would use a lacZ reporter to to find regulatory genes that would, for example, that would for example control the expression of a desired virulence gene. So, let's say I set up a transposon carrying a promoterless lacZ gene and an antibiotic resistance marker such as Knr. But how could I use this set up, specifically, to locate genes that would respond to specific conditions or signals? Suppose the genes that are being sought are expressed at high levels only under low…

Hello! I am a student of bioinformatics and I am about to start a project of mutational stability of proteins. As a base for comparision for my analysis, I am in need of proteins that have large datasets of experimentally determined ∆∆G values. In the most optimal case, the protein(s) shouldn't be coordinated to a metal-ion. I would be very thankfull for any pointers towards such proteins! I hope that any of you can help me (as google has not been my friend). Thank you very much!

How do I find the ribosome binding site of a phoA gene?

Is there a difference in efficiency of ligation between 5'-overhang and 3'-overhang? And do ligation products look like dsDNA? I don't understand how there are several ligation products when the PCR fragment digested by a restriction enzyme has the same sequence.

Lastweek, I synthesized cDNA from HEK293 cells and amplify RAD51-AP1 mRNA (BC016330.1) by PCR with the annealing temp of 65oC and with following primers: F-EcoRI: AT GAATTC A ATGGTGCGGCCTGTGAG; R-BamHI: AA GGATCC TCAGGTGCTAGTGGCATTTG and then I cloned this amplified fragment into P3XFLAG-CMV10 vector. However, the result of sequencing showed that I already cloned another gene which is CORO7-PAM16 mRNA. Please help me.

Is it possible for cells to make their own energy. I don't mean like in photosynthesis or chemosynthesis, I mean an autotroph that makes its own energy . In the sense that in a far away planet like Pluto that dose not have much gases or sunlight. Maybe, it's molecular structure is enough to give the organelles energy or something. maybe the first cell to evolve to this had enrgy and passed it on the the sister-cell's in miosis and the cell with a little bit of energy had an organelle that could make its own energy. Forgive me if this sounds stupid it just came to me.

I understand that the human body when performing Fatty Acid Synthesis can synthesize only until C16 (palmitate). However the ER has desaturases and elongases. I know that elongases are used to add double bonds, but do humans have desaturases to add even more Carbons to a double bond? If so, then this would mean that the human body can synthesize fats longer than C16? Lets take a hypothetical case and say that humans have both desaturases and elongases. Would it be feasible to add double bonds to palmitate via desaturases, then add more carbons to the chain via elongases and then add more double bonds via desaturases? This way, the resulting fatty acid will have an incre…

Dear fellow biochemists, I need some advice on Western Blotting, more specifically the use of certain protease inhibitors with the RIPA cell lysis buffer and protease inhibitor cocktail. A Millipore technical representative recommended my using "539134 | Protease Inhibitor Cocktail Set III, EDTA-Free - Calbiochem" as a good general protease inhibitor cocktail. I have another protease inhibitor cocktail which includes: AEBSF, bestatin, E-64, and pepstatin A (E.coli protease inhibitor cocktail) but it does not include the aprotinin or the leupeptin inhibitors. Is there a risk of proteolysis in a mammalian cell lysate without these latter 2 inhibitors? The specific cell ty…

Hi everyone, I´m on a Master program and next week I have to go to a Biotec company to solve a problem in just 1 week... I´ve been asked to characterise the human epidermal growth factor at least with two proteases. This protein is very small around 6kDa, and I´ve never done a peptide map of any protein. If anyone could guide me through the basics would be great. I need to understand what should I know about the protein and how to select the appropriate proteases, and how to actually see in a simple experiment the peptide map, like in electrophoresis. Thank you. Alvaro

i would like to know whether repeated freeze thawing of plasmid dna affect its intrgrity

Hi, I know the initial event of a pathway (i.e. inactivation of a kinase by dephosphorylation) and the end result (i.e. inhibition of apoptosis). However, I would like to figure out which proteins are involved in between the initial and the final events. The initial kinase has several targets and I would like to know exactly which proteins and pathway is modulated. Thanks!

This is a picture of the contamination that it appear in my cells last week. I don't know how to get rid of it and i have no idea of what it is. Can anyone help me? i really need to get this cells good as soon as possible to start an important experiment and all the ones we have are contaminated with this thing.

So I'm trying to get a more current answer to these questions because the only papers I can find are from over a decade ago and this field is moving too fast to take them at face value. So how many protein coding genes are there in the human genome? (HGP) How many of these do we have experimental evidence for? (UniProt?) and how many of these do we have an experimental structure for? (PDB?) Help and links would be greatly appreciated

I was wondering If my proposal is feasible, is it possible extract Tardigrade proteases and then clone them(the cDNA) into say E.coli then characterize them on the basis for using them industrially?

Hi there! I faced with problem, and I never worked with viruses before. Major aim - to dissociate virus capsid to the single capsomere units. Work will made with plant viruses and some bacteriophages. In the Internet I found a lot of papers about capsid extraction from native virions, but a bit information about capsomeres. Are the common methods to obtain capsomeres from virus particles? Best regards!