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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

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

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 ...


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 ...


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

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

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 ...


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 ...



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