Lab Isolation of Nucleic Acids 1 Flashcards
Steps in DNA isolation
- Release cells from sample
- Disrupt cell integrity & prepare cell free extract
- Remove debris from cell free extract
- Purify DNA from treated extract
- Measure quantity & quality of purified DNA
How does differential density gradient centrifugation make nucleated cells available
- WBCs settleand separate from other plasma components & cells
- These layers can be isolated & washed
How does differential lysis make nucleated cells available
- Uses differences in osmotic fragility of cells
- Incubation of whole blood in a hypotonic buffer will result in lysis of rbcs, before wbcs
- Rbcs more fragile
- Centrifugation leads to pellet of wbcs
How must tissue samples be prepared before DNA isolation can occur: fresh/frozen tissue
Must be dissociated before DNA isolation by:
* gringing frozen tissue in liquid nitrogen
* homogenising the tissue
* mincing the tissue w a scalpel
How must tissue samples be prepared before DNA isolation can occur: fixed embedded tissue
Has to be deparaffinated by:
* using xylene
* less toxic xylene alternatives
* tissue must then be rehydrated
How to prepare microorganisms for DNA isolation
- A culture of bacteria is grown & harvested
- Bacterial culture centrifuged
- Pellet of cells obtained
- Cells removed and broken to get a cell extract
- DNA purified from cell extract
- The DNA is concentrated
Organic isolation method of extracting pure DNA from cell extract
What is used to obtain purified DNA with this method
- High salt
- Low pH
- Organic mixture of phenol & chloroform
- RNAse can be added to degrade RNA
Organic isolation method of extracting pure DNA from cell extract
What does the addition of phenol & chloroform lead to & what is done after this
- Phenol & chloroform lead to formation of a biphasic emulsion
- This is centrifuged
- This settles the hydrophobic layer at the bottom & the hydrophilic layer at the top
- The upper phase containing DNA is collected
Organic isolation method of extracting pure DNA from cell extract
How is DNA precipitated after being collected after centrifugation
- DNA precipitated using ethanol or isopropanol in high conc of salt
- This is rinsed to remove excess salt
- DNA then dissolved in buffer
Inorganic isolation method of extracting pure DNA from cell extract
Why may this inorganic method be chosen rather than the organic method
Safety issues with phenol
Inorganic isolation method of extracting pure DNA from cell extract
Steps of this method
- Uses low pH & high salt conditions to selectively precipitate proteins
- This leaves DNA in solution
- DNA precipitated in alcohol
- DNA pelleted
- DNA resuspended in buffer or water
Solid phase isolation
Prep of samples for isolation begins with what?
Begins with cell lysis & release of nucleic acids
Solid phase isolation
How does the DNA adsorb to solid matrix
The cell lysate in high salt buffer is applied to the column & DNA adsorbs onto the solid matrix
Solid phase isolation
What happens after adsorption & washing
After washing the immobilised DNA is washed with buffer and the DNA is eluted in a specific volume of water or buffer
RNA isolation
Why must RNAse enzymes be eliminated before isolation of RNA
They are small proteins that can renature after autoclaving & become active
