Today we'll talk about plasmid DNA purification and quantitation.
By the end of this video you should be able to: list the main steps of plasmid isolation
and purification using alkaline lysis and silica adsorption, respectively. Describe
the importance of each step, use a micropipette to deliver a specific volume,
evaluate the purity of your DNA sample and calculate DNA concentrations.
The goal of our experiment is to end up with purified plasmid DNA.
In order to do that we're going to have to isolate the plasmid from the bacterial cell
where it was made, and all of the components like proteins, genomic DNA, and the lipids
that make up the cell.
We'll start our experiment with an overnight culture of bacteria which we'll centrifuge
to form a bacterial cell pellet.
We’ll remove the culture media and then we're going to resuspend the bacterial cell
pellet by repeatedly pipetting up and down.
We want to make sure that we completely resuspend the pellet, otherwise we'll have inefficient
lysis and a drastic reduction in the amount of plasmid DNA recovered.
Next, we will add our lysis buffer, if we're using a color indicator we'll see that the solution
will turn blue.
We want to rock the tube until it is completely blue which will indicate that we've lysed
all the bacterial cells.
We're using an alkaline lysis buffer, so that's going to raise the pH inside the tube.
The lysis buffer also contains a detergent, SDS, and together with this will have several
effects.
First, it’s going to lyse the bacterial cell, which is going to release all of the
cellular components.
The high pH is also going to cause our DNA strands to separate and no longer base pair.
It's important to remember that the DNA strands will still be physically associated with each
other even though they are not base paired.
That's true for both our plasmid DNA and our genomic DNA.
Additionally, the high pH is going to cause our proteins to denature and unfold.
In our next step, we're going to neutralize the pH with a weak acid.
Now that the pH is neutral again we're going to see a couple things happen.
First our DNA strands are going base pair again.
In the case of plasmids, which are relatively small, this can happen efficiently in a short
time.
In contrast, genomic DNA is not going to be able to efficiently base pair.
As a result, the genomic DNA, proteins, and lipids will all aggregate into a fluffy white
precipitate, which you’ll be able to see in the now colorless solution in your tube.
The aggregated material is insoluble, which allows us to separate it from the soluble
plasmid DNA with a centrifuge.
We have now isolated our plasmid DNA but we still have some contaminants, as well as a
lot of different ions that came along with our different buffers.
So before we can use our plasmid DNA we want to remove all of these contaminants.
We'll be doing that with a spin column that has a silica matrix.
Under high ionic strength the silica will selectively bind to DNA.
Once the DNA is bound, we can wash the column until we have removed all of our contaminants.
Once we're ready to elute, we will switch to a buffer that has low ionic strength.
Under these conditions the DNA will release from the silica matrix and we'll end up with
our purified plasmid DNA. So now that we have our purified plasmid DNA
we want to know two things about it.
We want to know the purity and concentration of the DNA.
Both of these questions can be answered with a spectrophotometer.
In our case we'll be using an Nanodrop spectrophotometer which has the advantage of only needing small
volumes of sample.
Since we're using small sample volumes, we need to make sure that we pipette accurately.
To do that we'll first need to select which micropipette to use.
Shown here are four different micropipettes.
They are referred to as P and the maximum volume they can accurately pipet.
For example, a P200 micropipette can pipet a maximum of 200 ul.
When selecting which micropipette we should use, always use the micropipette with the
smallest volume range that is appropriate for your desired volume.
For instance, a P10 and P20 could both be used to pipet 5 ul but P10 is the better choice
since it will pipet the smaller volume more accurately.
If we wanted to pipet 100 ul, which pipet should we choose?
While a P1000 could be used to pipet 100 ul, the best choice would be a P200.
Once we’ve selected our micropipette and are ready to pipet, we want to start by pushing
the plunger down to the first stop position.
You'll know you're there when you feel pressure.
Now with the plunger depressed, you can put your tip into your liquid.
You want to stay near the top of the liquid because if you go too far down you can end
up with liquid stuck to the side of your tip, which is going to affect the accuracy of your
pipetting.
Once you have your tip placed you can slowly release the pressure on your plunger, which
is going to pull liquid into your tip.
When you are ready to dispense, you will place your tip on the sidewall of your new tube.
You will push down to the first stop again, and then push even further to what's called
the second stop.
That's going to make sure that you've removed all the liquid from your tip.
After pipetting something especially viscous you might want to again release the plunger
and pull liquid back up and press down to the second stop again to really rinse your
tip.
The nanodrop has a little pedestal, shown here zoomed in, and we want to make sure that
when we pipette we end up with our liquid on top of this pedestal.
We will then bring the arm of the instrument down, press down, and then release.
When we're done we should have a column of liquid formed.
Before you measure your sample, you want to make sure that you have this liquid column
formed otherwise you will have an inaccurate reading.
The software will generate an absorbance spectrum after measuring our sample.
Shown here is spectrum that represents the absorbance of pure RNA, DNA, and protein.
We can see in pink the DNA and in green the RNA have a maximum absorbance at 260 nanometers.
In contrast, protein absorbs maximally at 280 nanometers.
To assess the purity of our samples we divide the absorbance at 260 by the absorbance at
280.
For pure DNA, we would expect a ratio of 1.8, for RNA we would have a ratio of 2, and with
protein we'd have a ratio of 0.8.
For DNA purification, the most common reason for a deviation from the ideal ratio is due
to chemical contaminants, which will give a lower ratio.
We can also use the absorbance at 260 to determine the concentration of our plasmid DNA.
One unit of absorbance at 260 corresponds to 50 micrograms per milliliter of DNA.
This constant has been determined empirically to be true for all double stranded DNA.
If our plasmid DNA is very concentrated, we may want to dilute it to get an accurate reading.
The dilution factor is calculated by dividing our final volume by our initial volume.
For example, if you had 10 ul of a DNA solution and you added 90 ul of water to it, you would
have a final volume of 100 ul.
Since the volume of our DNA solution has increased, the concentration of our DNA has decreased.
By dividing the final volume by the initial volume, we get a dilution factor of 10, meaning
that our DNA concentration is now 10-fold lower than when we started.
So to determine our concentration we take the absorbance at 260, we multiply it by 50,
and then multiply by our dilution factor to end up with the final concentration of our
DNA.
So by measuring the absorbance of our sample at 260 and 280 nanometers, we can get both
the concentration and the purity of our sample.
Now at the end of this video you should be able to list the main steps of plasmid isolation
and purification, describe the importance of each step, use a micropipette to deliver
a specific volume, evaluate the purity of your DNA sample, and calculate DNA concentrations.
Thanks for listening.