Do you have a short region of a DNA molecule that needs to be copied?
Well good news! I'm Alyssa, Senior Quality Control Scientist at Addgene
and we're going to show you how to do just that,
using a standard polymerase chain reaction.
Polymerase Chain Reaction (or PCR)
is an in vitro method that allows
scientists make tons of copies of a single short region of a DNA molecule,
like a gene, using a specific type of DNA polymerase.
From that single copy of DNA,
known as the template, thousands of identical copies can be created,
using a simple set of reagents and basic heating and cooling cycles.
PCR requires two unique and single stranded
DNA oligonucleotide primers which anneal to
the regions upstream and downstream of the DNA segment to be amplified.
To synthesize new DNA strands from the primer the PCR reaction requires free
nucleotides or dNTPs, and a DNA polymerase which adds these nucleotides
to the growing DNA strand polymerase is used in PCR can withstand many heating
and cooling cycles. In this video we will use tack polymerase.
Overall PCR relies on repeated cycles of DNA denaturation at high-temperature,
annealing of primers to the single-stranded DNA,
and synthesis of new DNA by the TAC polymerase
to create several micrograms of the target DNA from just a few nanograms of template DNA .
Once the PCR is complete the product can be verified based on
size by gel electrophoresis.
So now that you have a basic understanding of what
PCR is and how it works let's get to it!
To start the process you'll need some materials.
Get some thin walled PCR tubes,
an ice bucket,
and some agarose gel (but you can grab that a little later).
For each of the reactions you will need a
forward primer, a reverse primer, dNTPs, template DNA, Taq polymerase, buffer and water.
And of course you'll need a PCR machine,
which will do the heating and cooling steps for you.
Before you can begin your experiment you'll need to
design and order primers.
These are strands of nucleic acid that are the
starting points for DNA elongation and synthesis.
These primers hybridize and are reverse compliments
to both the ends of the DNA region you want to amplify.
You'll want your five-prime and three-prime primers to be designed to have similar
melting temperatures. If you need some help designing primers check out
Addgene's PCR cloning protocol.
Do you have everything you need? Great! Time to get started.
When you're ready to do your PCR head into the lab.
First, place the thin-walled PCR tubes on ice
Now you need to set up a 50 microliter PCR reaction mix.
For that you'll need the listed items.
Make sure you keep all of your reagents on ice during this step and throughout the protocol.
If you're doing multiple PCR reactions you can same time by creating what's known as a master mix.
The master mix the master mix contains the reagents common in all of your reaction tubes.
For example the water, Taq, and dNTPs.
It'll minimize the number of small volumes you need to pipette.
You can purchase a master mix with Taq polymerase included,
or you can check out our lab tip video if you're interested
in creating a master mix yourself.
Now you're ready to start the PCR process.
Place those reaction tubes into the PCR machine and set up the PCR steps.
The PCR machine will lower and raise the temperature for a set period of time
based on the program you enter
The first step in the PCR is the initial
denaturation for 2 minutes at 94 degrees Celsius.
This heats the double-stranded DNA template to the point where the strands start to naturing breaking the
hydrogen bonds between the nucleotide base pairs.
Following the initial 2 minutes, you want to set another denaturation step for 30 seconds at 94 degrees Celsius.
Just remember if your DNA sequence is GC-rich
you'll need to increase the time of the denaturing.
Now you want to lower the temperature to 5 degrees Celsius below the primers melting temperature for 30 seconds.
At this temperature range the forward and reverse primers are stable
enough to anneal to each of the single-stranded DNA template strands.
The DNA polymerase is also stable enough to now bind to the primer DNA sequence the
The last step is to extend the DNA at 72 degrees Celsius.
The extension step should be one to two minutes per kilobase of product
but can depend on what type of polymerase you are using.
72 degrees Celsius is right within the optimal temperature range for the Taq polymerase
enabling the DNA polymerase to synthesize and elongate the new target
DNA strand accurately and rapidly.
This denaturation annealing and extension is
repeated for 25 to 30 cycles.
Finally set the PCR machine to perform a final extension
for five minutes at 72 degrees Celsius.
This will help fill in any protruding ends of the newly synthesized strands
Since you've got some time you can prepare your agarose gel, now.
You'll also have a bit of time to yourself I'll see you in a couple hours!
Is your final cycle complete?
Now you can run two microliters of your PCR product on a gel to check size and concentration of the PCR product.
Check out our text protocol and videos about gel electrophoresis if you need any help.
Is your reaction not working?
Try adding one microliter of 25 millimolar magnesium chloride,
and/or one microliter DMSO, to each reaction.
Ideally you'd perform a reaction with each separately and a third with both together.
You can also play around with the annealing temperatures.
Lowering the temperature might give you more products but can also result in nonspecific
binding of the primers to the template.
Congratulations! You've just completed a successful PCR routine!
Thanks for watching. Check out our other protocol videos and leave a comment
below to let us know what videos you'd like to see or to tell us how we can improve
Visit addgene.org/protocols for the complete list of our protocols.
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