Okay, today we're going to go through a technique
known as PCR or Polymerase Chain Reaction.
This is a technique where DNA is amplified using known primers against a specific piece of DNA.
The DNA will be amplified by the Polymerase Chain Reaction
and the products of the reaction can be seen using agaros gel electrophoresis.
The gel is viewed under UV light and you can run markers of known size of base pairs,
and this can determine the size of the DNA which has been amplified.
Okay, there are several reagents
which are required for producing the master mix for the reaction.
The first reagent is buffer, which provides an optimum solution conditions
for the reagents to work.
This is added to your reaction tube.
The next reagent is magnesium chloride.
Between reagents always make sure you change the tip so you don't contaminate your reagents.
Magnesium chloride will be added.
Pipettes are used to ensure that you get the correct volume of each reagent.
As you add the reagent to your master mix,
you tick off your working list to make sure that you've know you've added your reagent
and that you don't add double the volume that's required.
Okay, so the next reagent will be magnesium chloride
and magnesium chloride helps the enzyme to anneal to the DNA strand.
The enzyme used is taq polymerase, which is added at the end
and magnesium chloride has been added.
Each volume of reagent is known on your worksheet,
and the pipette is adjusted to get the correct volume to add to your working master reagent.
Okay, the next is reagent is the forward primer,
and the primer is a piece of DNA which corresponds to the piece of DNA
that you're actually looking for, and the next reagent is the reverse primer.
This is added to your master mix.
As you can see, all agents are kept on ice.
The next reagent to add are the di-nucleotide triphosphates,
and these are the bases which will be used by the enzyme to synthesize
the new DNA in the amplification process.
After the di-nucleotide triphosphates have been added,
a volume of distilled water is added to the master mix,
and the final reagent to be added to the master mix is the taq polymerase.
Taq, meaning thermus aquaticus, which is the bacterium from where the enzyme
has been obtained,
and that's added to the master mix,
and that's ready to use and that's ready to be added to the sample.
Okay, so now we have our four tubes ready with the master mix present,
and all that remains is to add our DNA sample to each tube.
The tubes have been pre-labeled beforehand and
the scientists just checked the tube to make sure she was adding the correct sample
to the correct tube, which is vital when you're doing any assay.
And the sample is mixed well with the master mix reagent
sample three and a fresh tip is used each time for each new sample.
So now the sample has been added to the reagent,
and the next step will be the thermocycling of the DNA
to allow the polimerase chain reaction to occur.
Once the samples are prepared, they're then placed in the thermal cycler.
The lid is closed and the cycle is started,
and the cycler is pre-programmed to reach certain temperature for a certain period of time.
The initial temperature is ninty five degrees for five minutes,
and this allows for complete denaturation of the DNA,
and then the double stranded DNA separates and the primer attaches once it's cooled
to fourty five to sixty degrees.
This is where the annealing of the primer and the single stranded DNA occurs.
The recycler heats again to seventy two degrees C,
where the DNA polymerase amplifies the DNA,
and then it is heated again to ninty five degrees to separate denature the double stranded DNA,
this takes place over thirty cycles,
and the final extension is at seventy two degrees centegrate for five minutes.
Once the cycling is complete,
the samples are removed from thermo cycler and placed on ice,
and they're now ready to be run on an agarose gel,
or they can be placed in the freezer to be stored until they're ready to be run on an agarose gel.