DNA EXTRACTION AND
POLYMERASE CHAIN REACTION
TECHNIQUE
DNA EXTRACTION
Now that you’ve collected samples of bacteria from the biofilms it is
time to extract the DNA from the cells to identify the organisms.
DNA extraction involves several steps and
multiple tools. Here is a simple explanation
of the basic steps.
Step 1: The cell walls and membranes of
the bacteria must be broken apart. The cell
wall is made out of protein and the cell
membrane is made out of molecules called
lipids (similar to grease on your dirty
dishes).
Q: What do you use to remove grease from
dishes?
A reagent called a lysing buffer is added to
“lyse” or break the cell membranes, and
you guessed right, it is just like the soap
you use to wash dishes.
Another substance called an enzyme works
like a pair of scissors to break up the
proteins in the cell walls.
Step 2: Once the cells are broken apart the
DNA can be more easily extracted.
Fun facts:
*DNA is a negatively charged molecule
*Most proteins are positively charged.
The samples are incubated or heated aid in
the process of breaking up the proteins.
Many molecules do not like high
temperatures and the heat causes the
molecules to break into smaller pieces.
FUN FACT:
* The heating process also breaks up the
enzymes you added to destroy the cell wall.
This is helpful in removing the unwanted
material in the sample, smaller pieces are
easier to filter.
After incubation, the samples are
filtered using a special column that will trap
the DNA but allow all the other stuff we
don’t need to pass through.
Step 3: Once the samples have been
incubated, a centrifuge is used to
pull the liquid through the special
filter.
This is done a couple of times using
different solutions.
The first time the centrifuge is used,
a solution is added to pull
everything but DNA through the
filter.
The second time the centrifuge is
used, a solution is used to pull the
DNA out of the filter and into a new
container called a micro centrifuge
tube.
Now we are ready to make copies of
the DNA using PCR.
Polymerase Chain Reaction (PCR)
The final step before sequencing is to
make a lot of copies of our DNA sample so
that we have enough for the sequencing
machine to detect and read.
Polymerase Chain Reaction or PCR allows
us to target specific DNA segments and
quickly make copies.
View this short video to see how PCR
works.
Now try this:
Visit this site to carry out a virtual bacteria
identification lab.
Answer the questions on the next slide as
you complete the virtual lab.
Virtual Lab Questions
• What is the first step in extracting DNA from the bacteria? What part of the cell is involved and what are the chemicals and processes used
to facilitate this?
• What are the four nucleotides added to the master mix? Why are these important?
• What are the positive and negative controls used in the PCR process? What is the function of a positive and a negative control?
• Summarize the steps and note the temperatures and molecules involved:
Step 1
Step 2
Step 3
• During sequencing how are the A, C, T and G’s identified?