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DNA Techniques Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


Mini Prep

Mini Prep - DNA Techniques

Mini Prep - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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  #1  
Old 11-21-2007, 09:09 PM
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Default Mini Prep



Hello everyone i need help understanding why?
when carrying out the min prep what is the purpose of using solution 1, 2 and 3, why do we use phenol/chloroform? and in the last few steps why do we use ethanol?
solution 1 is:
stock solution volume final concentration
40% sterile glucose 2.27 ml 50 mM
0.5 M EDTA, pH 8 2.0 ml 10 mM
1M Tris-HCl, pH 8 2.5 ml 25 mM
sterile ddH2O 93.23 ml
Total: 100.0 ml

Use all sterile stock solutions. Store at 4 degrees C.

Solution 2 is:
stock solution volume final concentration
1N NaOH 2.0 ml 0.2 N
10% SDS 1.0 ml 1%
sterile ddH2O 7.0 ml
Total: 10.0 ml

Prepare fresh solution prior to use.

solution 3 is:
stock solution volume
5 M KOAc 60 ml
glacial acetic acid 11.5 ml
ddH2O 28.5 ml
100 ml

Filter sterilize. The resulting solution is 3 M potassium and 5 M
acetate and has a pH of about 4.8.

THANK YOU
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  #2  
Old 11-26-2007, 04:42 PM
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Default Re: Mini Prep

Solution 1 is TE buffer-- which may inhibit enzymes, but simply keeps everything stable--no real action
Solution 2 is obviously your lysis solution (with 10% SDS), it will disrupts membranes, micelles, protein-lipid pairs
Solution 3 is your salt--Acetate pairs with DNA to form a precipitate.

Phenol:Chloroform is commonly used in Nucleic acid separation from proteins and lipids.
Alcohol (Ethanol/Isopropanol) is used to dehydrate the DNA so it forms a pellet, and graded ethanols can be used to wash excess salts from the DNA pellet.
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Old 11-26-2007, 10:55 PM
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Default Re: Mini Prep

phenol:chloroform extraction is used to isolate DNA from proteins and cellular debris. Phenol and chloroform are organic solvents and therefore interact and trap hydrophobic entities (ie hydrophobic portions of denatured proteins). The interphase which is observed following treatment with these solvents, results when the hydrophilic portion of proteins interacts with the aqueous phase. It is therefore important to avoid the interphase when recovering the DNA containing aqueous phase.

phenol cloroform extraction is carried out prior to ethanol precipitation which concentrates the DNA.
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