DNA Isolation in Bacteria and Yeast

DNA has the appearance of white mucus

http://biology.clc.uc.edu/fankhauser/Labs/Genetics/DNA_Isolation/dna_isolation_jpg/20_DNA_clump_P1082649.JPG

DNA Purification & Isolation from Bacteria and Yeast

The preparation of DNA extraction should begin 36 hours in advance of the laboratory period. For spooling the DNA, the rods used are important for not to break the chain. It is possible to mix the ethanol and water to get more DNA to precipitate. The solution must be waited for 10 or more minutes for the DNA to appear.

The method in mixing the solution (especially without foaming when the detergent is added) is also important to get the whole segment without fragmentation. The detergent and heating (at 55 60C) dissolve the fats in the cell walls of the bacteria, thus freeing the DNA. Since DNA is not soluble in alcohol, when alcohol is added to the mixture, all the components of the mixture, except the DNA will be trapped in the solution whilst the DNA precipitates out of the alcohol layer.

DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis.

Three basic and one optional step 1. Breaking the cells open cell disruption or cell lysis By grinding the sample 2. Removing membrane lipids By adding a detergent 3. Removing proteins by adding protease (optional) 4. Precipitating the DNA with alcohol (usually ethanol or isopropanol)

What is a TE Buffer?

A buffer used in procedures involving DNA or RNA extraction. TE is derived from T Tris : A pH buffer that keeps the solution at a defined pH EDTA Ethylenediaminetetraacetic acid, which is a colorless, water-soluble solid. TE Buffer includes 10 mM Tris, bring to pH 8.0 with HCl 1 mM EDTA

EDTA further inactivates nucleases, by binding to metal ions required by enzymes.

http://openwetware.org/wiki/TE_buffer http://en.wikipedia.org/wiki/File:Ethylenediaminetetraacetic.png

DNA Isolation From Yeast Yeast has a thick, rigid structure. The usual yeast DNA preparations are contaminated with RNA. The procedure includes 1. 2. 3. 4. 5. Preparation of enzymatic digestion of the cell wall Deproteinization and extraction of lipids Enzymatic digestion of RNA Elimination of polysaccharides by centrifugation or digestion Follows, the DNA separation by selective isopropanol precipitation.

Methods in cell biology: Yeast cells David M. Prescott

A very rapid method for extracting DNA from bacteria and yeast Involves the extraction of genomic DNA from gram-negative bacteria, gram-positive bacteria and yeast Bacteria or yeasts are lysed directly by phenol The supernatant is extracted with chloroform to remove the traces of phenol. The supernatant contains DNA that is suitable for molecular analysis such as PCR, restriction enzyme digestion and genomic library construction.
http://www.mendeley.com/research/dna-extraction-fungi-yeast-bacteria/#page-1 http://www.ncbi.nlm.nih.gov/pubmed/16369876