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14

The role of the salt is to neutralize the charge of the DNA's sugar phosphate backbone. This makes the DNA less hydrophilic (less soluble in water). Ethanol has a lower dielectric constant than water so it's used to promote ionic bonds between the Na+ (from the salt) and the PO3- (from the DNA backbone) causing the DNA to precipitate.


7

The protocol I used in my genetics lab course was alkaline lysis followed by ethanol precipitation similar to this one. Nothing terribly toxic or requiring a fume hood (at least at the small volumes used). The more practical, reliable method is to use a miniprep kit (usually spin columns) from one of several suppliers (Qiagen, Promega, Bio-Rad, etc.) which ...


6

For the impatient, I'm going to say that probably if you only have a regular home freezer, its unlikely that you will be able to do this. If you have access to a -80C or liquid nitrogen storage its possible, but also less likely. Cloning an animal typically starts with the transplantation of a nucleus into an egg cell whose nucleus has been removed. So ...


6

In my experience, the P:C:I method will get you higher yields, and is a bit more simple (in steps and chems involved), but as @Mad Scientist has said, phenol use might be an issue. It depends on the age of your students.


6

Yes, you must fragment the genome in order to insert it into a vector for cloning; you can't "insert" the whole 5 Mbp genome of E. coli into a vector. It's difficult to transform cells with huge plasmids, 2-20 kbp is an optimal range. In any case, if you want a clone of the whole genome, wait 30 minutes and the cell will happily oblige you. Most ...


5

I think the spin column kits are the way to go. As mentioned already, the benefits of the kits are that they are easy, safe, and (most importantly) how almost every actual lab does plasmid purification these days. The biggest criticism of the commercial kits is that you can get by easily without knowing anything about what is actually happening in the ...


5

Using spin columns, my professor and I extracted ~30 plasmids from ~30 samples in a few hours. It's easy, cheap, and it yields good quality plasmid. My sample was quantified by UV spec and it was at about 200ng/ul.


5

A dependable protocol for yeast DNA extraction I used to use was broadly similar to the protocols you cite but included an ethanol precipitation before and after the P:C extractions. The only expensive material was time. The general outline was: Alkaline lysis Ethanol precipitation RNase A treatment of resuspended DNA for 15 minutes Phenol:chloroform ...


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 ...


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 ...


3

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. ...


2

I've successfully used the Zymoprep kit, but only for smaller plasmids in 2-hybrid experiments. So their cell lysis enzyme/buffer system works well, but I'd guess that your 42kb plasmid is precipitating out with the genomic DNA. The standard Qiagen midi columns are capable of capturing large constructs. This has worked wonders for me when I was purifying ...


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 ...


2

Proteinase K activity is greatly increased by addition of denaturing agents like SDS or urea (Hilz et al., 2008), indicating that the denaturation of the substrates helps Proteinase K to degrade them. Increasing the temperature to 50°C will also unfold some proteins already, making it easier for the Proteinase K to degrade them. The proteinase K seems to be ...


2

In your linked wiki article: "An elevation of the reaction temperature from 37 °C to 50 - 60 °C may increase the activity (of Proteinase K) several times." The enzyme works faster at 50°C.


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 ...


1

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 polysaccerides and other ...


1

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 ...



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