DNA extraction

Where is the dna?

the nucleus

some in mitochandria

and some in chloroplast of plant cells

WHY PERFORM DNA EXTRACTION?

• DNA extraction is a routine method used to isolate DNA

from the cell’s nucleus, mitochondria, or chloroplast (only

in plants

Extracted DNA can be used in

• Polymerase chain reaction (PCR)

• Restriction enzyme digestion

• electrophoresis

• Fingerprinting

• Cloning

when to chose a particular technique?

• DNA can be isolated from any living organism using many different methods that require various chemicals.

• Which method to use depends on the sample type and DNA purity and yield required.

• There are a variety of DNA extraction protocols

• choose the one that is most suitable for your experiment.

• might require several testing and trials to find the write protocol for you.

TYPES OF PROTOCOLS

1. Solution-based (Chemical) DNA methods

2. Solid-phase (Physical) DNA methods

Solution-based (Chemical) DNA methods

a. Organic DNA extraction (uses organic solvents like phenol-

chloroform)

b. Inorganic DNA extraction (uses salts and detergents; includes

salting-out methods)

c. Proteinase K DNA extraction (digests proteins to purify DNA)

Solid-phase (Physical) DNA methods

a. Silica-Column Based Extraction (DNA binds to a silica membrane)

b. Magnetic Bead-Based Extraction (DNA binds to coated magnetic beads)

c. Paper-Based DNA Extraction (uses specialized paper to capture DNA)

DNA EXTRACTION STEPS

1)Lysis - Breaking open cells to release DNA

2)Precipitation - Separating DNA from proteins, lipids, and other cellular components

3)Purification - Removing contaminants such as proteins, RNA, and cellular debris

4)Recovery - Collecting purified DNA for use in downstream applications

STEP 1- LYSIS

The cells in a sample are separated from each other, often by a physical means such as grinding or vortexing (with or without liquid nitrogen).

• The sample is then put into a solution containing salt.

• The positively charged sodium ions in the salt help protect the negatively charged phosphate groups that run along the backbone of the DNA.

• A detergent is then added.

• The detergent breaks down the lipids in the cell membrane and nuclei.

• DNA is released as these membranes are disrupted.

• Both cell and nuclear membranes (and cell wall in plants) must be broken down.

STEP 2- SEPARATING DNA FROM PROTEINS AND OTHER CELLULAR DEBRIS

• To get a clean sample of DNA, remove as much of the cellular

debris as possible.

• Centrifuge to separate the solids from the dissolved DNA

• Further removal of cell debris and proteins by adding

proteases.

• Proteases are enzymes that digest proteins.

STEP 3- PRECIPITATING THE DNA WITH AN ALCOHOL

• Ice-cold alcohol (either ethanol or isopropanol) is carefully added to the DNA sample.

• DNA is soluble in water but insoluble in the presence of salt and alcohol.

• DNA precipitates out of the solution

• If there is lots of DNA, you may see a stringy, white precipitate

• Centrifuge to separate the DNA from the dissolved salts and sugars

• Discard supernatant and wash with Ethanol

• Dry the pellet

• Air dry: tubes open at RT for

~15min

STEP 4- RECOVERY

• Resuspended DNA in a slightly alkaline buffer (TE buffer,

pH 8.0) or nuclease-free H2O

• DNA pellets will not dissolve well in high-salt buffers.

• Small quantities (<25 ug) of precipitated plasmids or

restriction fragments should dissolve quickly upon

gentle vortexing or flicking of the tube.

• Larger quantities of DNA may require vortexing and

brief heating (5 min at 65°C) to resuspend.

• High-molecular-weight genomic DNA may require one

to several days to dissolve and should be shaken

gently (not vortexed) to avoid shearing, particularly if

it is to be used for cosmid cloning or other applications

requiring high-molecular weight DNA.

CONFIRMING THE PRESENCE AND QUALITY OF THE DNA

• For further lab work, it is important to know the concentration and quality of the DNA.

• Optical density readings taken by a spectrophotometer can be used to determine the concentration and purity of DNA in a sample.

• Alternatively, gel electrophoresis can be used to show the

presence of DNA in your sample and give an indication of

its quality.

STORAGE OF DNA

• DNA can be stored at 4 o C for extended periods,

however for long term storage, -20 o C is usually

utilized.

• Avoid repetitive freeze thawing of DNA, since this

can cause degradation.

• The storage of DNA at 4o C is better than -20 o C

and storage at room temp dried with stabilizer is

even better (lee et al. 2012)