This document provides the steps to isolate DNA from rat liver tissue. The key steps include: mincing fresh rat liver tissue and lysing the cells using SDS and EDTA solutions; extracting the lysate with chloroform-isoamyl alcohol to remove proteins and lipids; precipitating the DNA from the aqueous phase by adding ethanol. The isolated DNA is then dissolved in SSC buffer for storage. The overall goal is to extract pure DNA from rat liver cells while minimizing degradation.
This document provides the steps to isolate DNA from rat liver tissue. The key steps include: mincing fresh rat liver tissue and lysing the cells using SDS and EDTA solutions; extracting the lysate with chloroform-isoamyl alcohol to remove proteins and lipids; precipitating the DNA from the aqueous phase by adding ethanol. The isolated DNA is then dissolved in SSC buffer for storage. The overall goal is to extract pure DNA from rat liver cells while minimizing degradation.
This document provides the steps to isolate DNA from rat liver tissue. The key steps include: mincing fresh rat liver tissue and lysing the cells using SDS and EDTA solutions; extracting the lysate with chloroform-isoamyl alcohol to remove proteins and lipids; precipitating the DNA from the aqueous phase by adding ethanol. The isolated DNA is then dissolved in SSC buffer for storage. The overall goal is to extract pure DNA from rat liver cells while minimizing degradation.
This document provides the steps to isolate DNA from rat liver tissue. The key steps include: mincing fresh rat liver tissue and lysing the cells using SDS and EDTA solutions; extracting the lysate with chloroform-isoamyl alcohol to remove proteins and lipids; precipitating the DNA from the aqueous phase by adding ethanol. The isolated DNA is then dissolved in SSC buffer for storage. The overall goal is to extract pure DNA from rat liver cells while minimizing degradation.
The DNA collected was dissolved in 15ml of solution B, in a boiling tube.
Solution B contains ribonuclease which is an enzyme used to break down RNA so that contaminating RNA is removed. The tube was incubated at 37C for 30 minutes, so that the RNase is able to work at optimal temperature. After 30 minutes, a solution of 5% (w/v) sodium dodecyl sulphate (3ml) was added so that the final concentration is 1% (w/v). The sodium dodecyl sulphate eliminates and precipitates RNA.
The solution was transferred into a 250ml conical flask and extracted (Shaking with mechanical shaker for 30 minutes) with an equal volume of chloroform. Chloroform plays a role in denaturing proteins and dissolving lipids. The solution was then centrifuged at 2500 r.p.m. for 5 minutes.
The supernatant was removed and transferred into a 250ml conical flask. To the supernatant, an equal volume of solution C was added. Solution C contains phenol which is a strong denaturing agent for proteins, the contaminants aggregate into the organic phenol phase and the DNA remains in the supernatant. The solution was put into the shaker for 20 minutes and then centrifuged at 2500 r.p.m. for 15 minutes.
The supernatant which contains the DNA was removed and transferred into a beaker. One volume of cold ethanol is added to the supernatant with stirring. The ethanol dehydrates the DNA so that it is easily removed from the solution. The cold temperature prevents the DNA from breaking apart. The DNA was collected on the glass rod and rinsed with 3ml 96% ethanol.
The spooled DNA was dissolved in SSC. SSC is used as a hybridisation
buffer to control stringency during the washing step. It also increases ionic strength so that DNA precipitation increases.
A sample of 5g of fresh rat liver was taken into a weighing boat.
This liver was extracted from rats which have ingested only water 24 hours prior to the extraction. The rats were not fed food, since the sample would become glycogen contaminated.
The liver sample was run under tap water to remove the blood, and was blotted dry with filter paper to remove excess water. The liver was transferred to a mortar where it was minced to a paste form using scissors, and then pulped with a pestle. This pulp was then transferred into a 200ml beaker.
To the liver tissue in the beaker, 8 volumes (roughly 40ml) of solution
A was added. The 0.1M NaCl serves as an osmotic stabilizer. The 0.1M EDTA is used since it chelates divalent ions such as Mg 2+, most DNases require Mg2+ as a cofactor thus EDTA chelates these ions to inhibit DNases. Therefore, degradation of the DNA is minimised. SDS plays a role in solubilizing and denaturing of proteins as well as lysing the lipid membrane component of the cell.
The solution was transferred into a dounce homogeniser. This is a high
pressure system that is used to break up cells. From the homogeniser, the solution was transferred into a 250ml conical flask. To the flask, an equal volume (40ml) of chloroform-isoamyl alcohol is added, and stoppered with foil. Chloroform-isoamyl alcohol is an organic solvent used to precipitate proteins, lipids and nucleases, leaving the DNA unharmed. The flask was put into a mechanical shaker to shake for 30
The solution was transferred into a centrifuge tube and centrifuged at
2500 r.p.m. for 15 minutes. Centrifugation is used to separate fluids, based on their density. The supernatant aqueous phase was recovered into a 250ml conical flask. The supernatant was reextracted as above with chloroform-isoamyl alcohol, again shaking for 20 minutes and centrifuging for 15 minutes.
The supernatant was recovered into 200ml beaker. To the supernatant,
with stirring, an equal volume of cold ethanol was added. The ethanol dehydrates the DNA so that it is easily removed from the solution and coils around glass rod while stirring. The cold temperature prevents the DNA from breaking apart. The DNA is collected on the glass rod.
