Transcript DNA Extraction Lab

DNA Extraction Lab
Step by step procedure that weakens
the outer boundaries of a cell and lyses
it to release the DNA for future study.
DNA Extraction Process involves four parts:
1. Put the cells into a solution.
2. Use enzymes to hydrolyze the cell wall.
3. Use a detergent to break apart the cell
membrane.
4. Use 95% ethanol to take out the DNA from
the lysate.
The above is the bacteria used in the DNA extraction lab. Note its
Spherical appearance and the color after gram staining. The gram stain
specifically stains the cell wall. According to this picture, is it gram
positive or negative?
Check out the Bacteria Power Point to find out about Gram Positive and
Negative Staining…..
Extraction Solutions:
1. Bacterial Suspension Medium – used to make
a solution of the cells
2. Lysozyme – enzyme that hydrolyses specific
bonds that hold the cell wall
3. SDS – a detergent that lyses the cell by
removing the lipids from the cell membrane
By adding these solutions to a test tube of
bacteria, a lysate is made that contains DNA.
The Lysate
Contents of the lysate after shaking,
rotating and inverting the tube:
1. DNA
2. Nucleases
The lysate is then placed in a hot water bath set at 60-65 degrees Celsius. Why?
Enzymes denature at high temperatures. Nucleases are enzymes that digest DNA so
the hot water bath destroys the damaging nucleases in the lysate.
DNA Extraction Technique:
In this picture, the student used 95%
ethanol to cause the DNA to
precipitate out of the lysate. DNA is
soluble in the lysate but insoluble in
ethanol. By drawing the lysate up into
the alcohol layer, the DNA comes in
contact with the ethanol, thus,
becoming visible to the naked eye.
Answers to Lab Q’s
1.
The SDS removes the lipids from the cell membrane. Therefore, it
weakens it and disrupts it to release the contents of the cell along with
DNA.
2. Lysozyme-suspension is added before the SDS to break down the cell
wall. It hydrolyzes the bonds in the cell wall. Once the cell wall is
weakened, the SDS can disrupt the cell membrane.
3. Protein denature at 60 degrees celsius because it contains fewer
hydrogen bonds than DNA. DNA is composed of many nitrogenous base
pair and so, it has many more hydrogen bonds.
4. A denatured protein has lost its shape and therefore can no longer
function. Nucleases in this lab were denatured in the hot water bath.
6. DNA should attach to the spooling rod at the aqueous-ethanol interface.