16

As it turns out, you are not the first to have this idea. Prior research claims that it is, at least in principle, possible to conduct epidemiology of cancer by sequencing on wastewater DNA for biomarkers. The ethics of this approach also need not be problematic because narrowly targeted sequence amplification focused on biomarkers like SNPs will not ...


9

Firstly, A DNA sample obtained from a cell or whatever is never going to contain just the DNA sequence you want but will instead contain the entire genomic DNA, plasmids, RNA sequences and whatnot. By using PCR you can selectively multiply the amount of desired DNA sequence while the undesired sequences do not get copied. Keep the PCR going long enough and ...


5

The DNA solution has a fixed volume (e.g., 0.1 mL). To a suitable test tube is added 0.2 mL of buffer-saturated phenol. After closing the test tube the mixture is mixed well, typically using a vortex mixer, but possibly by careful, repeated inversion. The aqueous phase and the organic phase are barely miscible. The tube is centrifuged to speed up the ...


5

As already hinted in the answer by @jakebeal, the low concentration (high dilution) of human DNA in waste water would mean that we need huge sequencing depth in order to detect something. This is not the case when dealing with urine or feces. This is also not the case when sequencing bacteria in waste water, since we are dealing then with the strains that ...


4

There are a few critical steps (which sounds horrible if written together, but the method per se is robust and usually without problems): Resuspension of the pellet: Make sure it is really resuspended and not floating around as a big blob. If it is not resuspended properly your yields will go down dramatically. NaOH/SDS-Lysis: Don't lyse for too long as you ...


4

There are already many great answers to your question, however I thought I put my comments in form of an answer. The standard for DNA agarose gel is TAE and for the protein, it depends on the size of the protein and the gel type used! Some times MOPS works best and sometimes Tris-acetate works best. It really depends on the gel used and also the protein and ...


4

Grossly, it does not matter what buffer you use. It is the pH that matters. For DNA electrophoresis EDTA is added in order to chelate divalent cations that serve as cofactors for nucleases. Tris is the base of the buffer and is used to set pH. Along with Tris one can use Boric acid, Acetic acid or phosphoric acid for adjusting the pH. The buffering range ...


4

The question which buffer for DNA is better is quite old. Both have their pros and cons and I list a few of them: TBE is a better conductor and is thus less prone for overheating the gel Borate is a powerful enzyme inhibitor, so if you want to apply enzymatic steps downstream, TAE is the better choice TAE gives a better resolution for large fragments TBE ...


4

I have had good experience using a lithium boric acid buffer from Faster Better Media. I use it for RNA gels, but it's advertised for DNA gels. I don't think it can do protein, but I've never tried it. I'm not an electrician, but higher conductivity may be the opposite of what you want. The lithium boric acid buffer claims to have less conductivity than a ...


4

For the precipitation you use different reagents: First you add salt under slightly acidic conditions to make sure that the DNA precipitates in a less polar environment. This is achieved by adding alcoholes like ethanol or isopropanol. It is commonly thought that a incubation at low temperatures enhances the results, but this is interestingly not true. ...


4

Guanidine HCL is a chaotropic salt. Chaotropic salts are critical for cell lysis and binding to the silica resin. Specifically, Chaotropes have two important roles in nucleic acid extraction Destabilize hydrogen bonds, van der Waals forces and hydrophobic interactions. Leading to destabilization of proteins (including nucleases). Macromolecular ...


4

From a computer science perspective, there's nothing at all special about DNA. It's stored as a simple ASCII text file consisting of repetitions of 4-15 different letters. DNA, the molecule, is a long chain of nearly identical smaller molecules (nucleotides) joined together. The nucleotides differ only in which of four possible bases (A,C,T/U or G) they ...


4

I've been trying to figure out something that works for some time. I have recently tried 3 cheap protocols using commonly available lab reagents. I was using buccal samples (0.2 ml of spit). 1) Boil the sample in Tris EDTA Buffer 2) NaOH Extraction 3) Direct N Lyse I compared them to a genomic DNA extraction kit (FAST DNA Prep - MP Biomedicals). I ...


4

Saliva buffer usualy provides stability at RT for month or even years. Genotyping companies like 23&me use collection kits like Norgen's (https://norgenbiotek.com/product/saliva-dna-collection-preservation-and-isolation-kit) which allows to store the sample up to 5 years. So my advice would be: get the spitting vial at your place, send it as regular ...


4

Purification and isolation of DNA bands by cutting them from agarose gel is commonplace (Lee et al., 2012). The purification step after excision of the band gets rid of most of the EtBr and other impurities. E.g., see the websites from Isogen and Sigma-Aldrich. Reference - Lee et al. J Vis Exp (2012); 62: e3923


4

No, you cannot pellet dissolved DNA with ultracentrifugation. Yes, you can recover a pellet with additional treatments, similarly to how you got it in the first place; only instead of your input being homogenized cells, or tissue, or extract, or whatever you used to obtain your DNA, it would now just be your aqueous solution of DNA. For instance, you may ...


3

It should be fine. Unless they have been contaminated with DNase, none of the components will damage or denature DNA. I've left purified plasmid DNA on the bench for weeks with no apparent degradation, so you should be okay (not that I recommend doing that on a regular basis). I would continue with the purification, according to your professor's ...


3

We had to do this frequently. What I would first recognize is that all of your current methods only work for at the small scale and that for you to purify mg quantities of DNA, you need to work with mg scale equipment. First of all, you should examine your "upstream" process. Are you doing your PCR at scale? Typically, we have scaled out by going with ...


3

As pointed out by aandreev, the greater the size of the insert relative to the flanks lower will be the recombination rate. This is because the donor DNA essentially becomes non-homologous. See the figures below:                 Figure1: Effect of insert size on recombination ...


3

The paper "Modified method for combined DNA and RNA isolation from peanut and other oil seeds." has a nice protocol for isolating DNA and RNA (which later can either be differentially digested or precipitated) from oil seeds for PCR. You can find the complete paper in the link above, the materials and methods sections says the following for the extraction ...


3

CTAB forms insoluble complexes with nucleic acids and can be used to selectively precipitate them from solutions, see this reference: The isolation of bacterial nucleic acids using cetyltrimethylammonium bromide (Cetavlon) When you add NaCl in a concentration between 0.4 - 0.7M, the nucleic acids stay in solution, while polysaccharides and other ...


3

It depends on the seed and fruit you're talking about. The amount of DNA in your crushed up sample of plant matter depends on only one thing--how many cells are in your sample. Look at this diagram of a kernel of corn (U of Indiana): There are different cell densities in each area--in the endosperm, the cells can be huge (and therefore have a lower density ...


3

It comes down to the size of genomic DNA (relatively huge) compared to that of plasmid DNA (rather small). Cell lysis for plasmid extraction involves an alkaline buffer that includes sodium hydroxide and SDS. Both plasmid and genomic DNA denature in this condition. When the alkaline reaction is neutralized with potassium acetate, the fairly short plasmid ...


3

Centrifuge the suspension in Cesium Chloride solution at a particular g value which causes the mitochondria to settle to the bottom of the centrifuge tube but leaves nuclear DNA forming a layer near the top of the centrifuge tube. This is since the nuclear DNA is less dense than the mitochondria. Using a needle syringe, extract a small part of the ...


3

Yes, normal DNA extraction protocols fragment genomic DNA. In my experience, extracted DNA tends to have a size distribution on the order of 10 - 20 kilobase pairs (kbp), independent of the protocol and source organism (assuming a chromosome size >> 20 kbp). This seems to be corroborated by what I can find online -- see the gel below from Successful ...


2

The units here are relative units of intensity. There may be about a picomole of probes on a microarray spot, but the units of intensity are not scaled to the precise concentration of DNA on the spot. There are many variables which make exact measurements of intensity difficult to translate into the number of RNA bound to a spot. The main one is ...


2

Here is the rough procedure that I've used to prepare cDNA before Extract RNA from desired tissue (select for mRNA using the polyA tail of mRNA) Conduct reverse transcription. This requires a primer complementary to the polyA tail to get the reverse transcriptase started (Edit:You could also use random primers to prime reverse transcription. This eliminates ...


2

To note: I have no personal experience with this protocol. I am forwarding this paper, passed on to me by a colleague There are many kits and methods available that don't rely on Qiagen. Or alternatively, if you ask them nicely, they do heavily discount their first kit you buy off them as a sort of trial (as they dont do samples). However, to answer your ...


2

It depends on what form your proteinase K is in. You may have a stock solution of some concentration, in which case you just add a specific volume according to $C_1V_1=C_2V_2$. Example: Let's say you want to prepare 5 mL ($V_2$) of 200 µg/mL ($C_2$) proteinase K from a stock of 1000 µg/mL ($C_1$). You're looking for the volume of stock solution to add ($...


2

EDTA carbohydrates, phenol all have absorbance near 230 nm. TRIzol reagent is a phenolic solution which absorbs in the UV both at 230 nm and ~270 nm Guanidine HCL used for most DNA isolations will absorb at ~230 nm also All these things will seriously inhibit your pcr, (and lower your ratio) especially the denaturing agents like guanadine. All these ...


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