text
stringlengths 3
503
| title
stringlengths 10
73
|
|---|---|
And let's say that one of these fragments contains a specific gene that we want to study. | Southern and Northern Blotting.txt |
The question is how do we identify that specific fragment that is carrying the gene and how do we isolate and separate that fragment to basically study it further? | Southern and Northern Blotting.txt |
Well, we have to carry out a process known as southern Blotting. | Southern and Northern Blotting.txt |
In southern Blotting we essentially use gel electrophoresis as we'll see in just a moment to basically identify and isolate a specific DNA fragment of interest. | Southern and Northern Blotting.txt |
So let's begin with step number one. | Southern and Northern Blotting.txt |
So we take our double stranded DNA molecule that contains the gene that we want to study. | Southern and Northern Blotting.txt |
Now we know what the sequence of nucleotides within that gene is before we actually carry out that process and we'll see why that's important in step three. | Southern and Northern Blotting.txt |
So we take the DNA, the double stranded DNA molecule, we expose it to specific restriction enzymes so that when they clean that DNA molecule one of these fragments will carry that gene of interest. | Southern and Northern Blotting.txt |
So let's suppose we break it down into five different fragments and these fragments differ in their size. | Southern and Northern Blotting.txt |
So we have the largest fragment of fragment A, we have the smallest fragment of fragment E and the fragment in between these fragments in terms of their size is fragment C. And that's the fragment, let's say, that contains the gene that we want to actually study. | Southern and Northern Blotting.txt |
So fragment C is that DNA fragment, that restriction fragment that we actually want to identify and then separate. | Southern and Northern Blotting.txt |
So let's move on to step two. | Southern and Northern Blotting.txt |
So we take these fragments, we essentially place them into a solution that denatures the double helix structure. | Southern and Northern Blotting.txt |
And so now in our solution we have these single stranded individual DNA molecules and now we take them and place them into a gel electrophoresis seven. | Southern and Northern Blotting.txt |
Now, if the DNA molecule isn't too large we can use the polyacrylamide gel. | Southern and Northern Blotting.txt |
But if the DNA molecule is very large then we have to use a gel that has larger pore size. | Southern and Northern Blotting.txt |
So we normally use anguarose gel. | Southern and Northern Blotting.txt |
So these are the two gels that we can basically use and the one that we use determines or which one we use is determined by the size of that initial DNA molecule. | Southern and Northern Blotting.txt |
Now, so we take our fragments and we place them into the gel electrofreeze setup. | Southern and Northern Blotting.txt |
And now the gel electrophoresis basically separates the DNA fragments based on size. | Southern and Northern Blotting.txt |
So the largest fragments, fragment A, will be all the way at the top because it will experience the greatest resistance, while the smallest fragment, fragment e, will be found all the way at the bottom because it does not experience a large resistive force. | Southern and Northern Blotting.txt |
And once we separate the five fragments based on size, we can then basically transfer that result onto a special polymer sheet that we can use more effectively. | Southern and Northern Blotting.txt |
And usually we use the nitrocellulose polymer sheet. | Southern and Northern Blotting.txt |
Now, once again, it's important to know that within these regions, these are no longer in their double stranded form, they exist as single stranded DNA molecules. | Southern and Northern Blotting.txt |
And that leads us directly into step three. | Southern and Northern Blotting.txt |
So in step two, we essentially separated these fragments based on size. | Southern and Northern Blotting.txt |
The next question is how do we identify which one of these bands contains that gene of interest? | Southern and Northern Blotting.txt |
So remember in the beginning I said that we have to know what that sequence of nucleotides is in that gene that we want to isolate. | Southern and Northern Blotting.txt |
And so what we do in step three is we build a DNA molecule, a DNA probe that contains a complementary nucleotide sequence that is complementary to that DNA fragment, the gene that we want to isolate found in fragment C. And when we build it, we radioactively label that DNA probe. | Southern and Northern Blotting.txt |
For example, we use radioactively heavy phosphorus atoms. | Southern and Northern Blotting.txt |
And what that will allow us to do is in step four, we're going to be able to use x ray autorediography to basically find exactly where that DNA molecule is. | Southern and Northern Blotting.txt |
So in step three, a specific restriction fragment of interest. | Southern and Northern Blotting.txt |
So this seed can be detected by creating and adding a radioactively labeled complementary DNA strand to that polymer sheet. | Southern and Northern Blotting.txt |
Since it is complementary to the gene of interest, it will hybridize with the fragment of interest. | Southern and Northern Blotting.txt |
So it will basically form a double stranded, form a double stranded helix. | Southern and Northern Blotting.txt |
So, to see exactly what we mean, let's take a look at the following diagram. | Southern and Northern Blotting.txt |
So in this diagram, it is before we added that DNA probe. | Southern and Northern Blotting.txt |
And so if we zoom in on this band, band C, we basically get this double stranded DNA molecule has been denatured. | Southern and Northern Blotting.txt |
And so these two individual single strands exist as single strands. | Southern and Northern Blotting.txt |
Now, when we add that DNA probe that has been radioactively labeled by, let's say, a heavy phosphorous atom, so what will happen is, because the sequence is complementary to this sequence here, the green radioactively labeled DNA probe will hybridize, will form a double helix structure with that single stranded complementary molecule. | Southern and Northern Blotting.txt |
And so now this has been radioactively labeled. | Southern and Northern Blotting.txt |
And notice this DNA probe will not form the same double helix with any of these other bands because the other bands don't have that complementary sequence. | Southern and Northern Blotting.txt |
So this molecule is a single stranded DNA fragment of interest that we want to actually detect. | Southern and Northern Blotting.txt |
And the green molecule is that radioactively labeled DNA probe that contains a nucleotide sequence that is complementary to that restriction fragment above that we essentially want to detect. | Southern and Northern Blotting.txt |
And once we carry out step three, then we can use the process of order radiography and that will allow us to pinpoint exactly where that fragment is. | Southern and Northern Blotting.txt |
And now we know if we go back to this setup, that this band C contains those fragments that we want to isolate. | Southern and Northern Blotting.txt |
And so we can take out the fragments, remove the other unwanted fragments, and now we have a pure solution that contains only the fragment, only that gene that we were actually interested in the first place. | Southern and Northern Blotting.txt |
So this process by which we can actually pinpoint, detect and then isolate that DNA fragment of interest by using our DNA probe is known as southern blotting. | Southern and Northern Blotting.txt |
But we also can repeat the same exact process with RNA molecule. | Southern and Northern Blotting.txt |
So if we have an RNA molecule that we actually want to isolate, we can use an RNA probe or radioactively RNA probe in the same exact process. | Southern and Northern Blotting.txt |
But if we're dealing with RNA, the process is known as north and blotting. | Southern and Northern Blotting.txt |
So in the same analogous way, we can conduct the same steps to separate and locate RNA fragments. | Southern and Northern Blotting.txt |
But instead of using the DNA probe, we use a radioactively labeled RNA probe in this process is known as north and Blotting. | Southern and Northern Blotting.txt |
So in southern blotting we essentially pinpoint, we detect and separate DNA fragments. | Southern and Northern Blotting.txt |
In northern blotting, we detect and separate RNA fragments and in western blotting we basically detect and isolate our protein fragments as we discussed in our lecture on purifying proteins via western blotting process. | Southern and Northern Blotting.txt |
Inside our body, we have many different types of exergonic and endergonic reactions. | Gibbs Free Energy and Spontaneity.txt |
Now, we know to basically calculate, to determine whether reaction is actually exergonic or endergonic, we have to calculate what the gibsfree energy is of that reaction because ultimately it's the gift's free energy value that tells us whether reaction is spontaneous or non spontaneous. | Gibbs Free Energy and Spontaneity.txt |
Now, the mathematical equation that allows us to actually calculate the magnitude of Gibbs free energy is this equation here. | Gibbs Free Energy and Spontaneity.txt |
So the gibsfree energy of that particular reaction under those conditions is equal to the Gibbs free energy under standard conditions when the concentration of products and reactants is equal to one molar plus this entire quantity. | Gibbs Free Energy and Spontaneity.txt |
So 2.33 times r t log q, where r is simply the gas constant, t is the temperature in Kelvin and q is the reaction quotient. | Gibbs Free Energy and Spontaneity.txt |
And that tells us the ratio of the concentration of products to the reactants. | Gibbs Free Energy and Spontaneity.txt |
Now, what exactly is the meaning of delta g? | Gibbs Free Energy and Spontaneity.txt |
Well, delta g tells us the amount of free energy that is produced or used up when a chemical reaction takes place under certain conditions. | Gibbs Free Energy and Spontaneity.txt |
So if a chemical reaction takes place and it releases gets free energy, that reaction will have a negative delta g value. | Gibbs Free Energy and Spontaneity.txt |
And such a reaction is said to be spontaneous exergonic. | Gibbs Free Energy and Spontaneity.txt |
And so this reaction releases useful energy that can be used to power other processes and reactions inside our cells and inside our body. | Gibbs Free Energy and Spontaneity.txt |
Now, if the delta g is positive, what that means is that particular reaction will be endergonic, non spontaneous, and a positive delta g means we have to input energy for that reaction to actually take place. | Gibbs Free Energy and Spontaneity.txt |
And in fact, inside our body, we can use exergonic reactions to produce energy and then that energy can be used to carry out undergonic reactions. | Gibbs Free Energy and Spontaneity.txt |
Now, what about when the delta g is zero? | Gibbs Free Energy and Spontaneity.txt |
Well, when the delta g is zero, that means our reaction has achieved equilibrium and this q value will become a k, the equilibrium constant. | Gibbs Free Energy and Spontaneity.txt |
Now, what about the delta g with this degree symbol? | Gibbs Free Energy and Spontaneity.txt |
What's the meaning of this quantity? | Gibbs Free Energy and Spontaneity.txt |
Well, this describes the free energy value of the reaction under specific conditions called standard state conditions. | Gibbs Free Energy and Spontaneity.txt |
And this describes conditions when the concentration of the reactors and products is equal to one molar. | Gibbs Free Energy and Spontaneity.txt |
Now, to see what we mean by that, let's take a look at the following graph. | Gibbs Free Energy and Spontaneity.txt |
So this energy graph basically contains the y axis, that's the Gibbs free energy. | Gibbs Free Energy and Spontaneity.txt |
And the x axis is the reaction progress. | Gibbs Free Energy and Spontaneity.txt |
And in this particular example, I used this chemical reaction. | Gibbs Free Energy and Spontaneity.txt |
So formic acid associates into the conjugate base and produces the H plus ion. | Gibbs Free Energy and Spontaneity.txt |
Now, let's suppose the concentration of this is one molar, and the concentration of these two products is also one molar. | Gibbs Free Energy and Spontaneity.txt |
So when this is the case, we see that when 1 mol of formic acid at a concentration of one molar transforms into 1 mol of conjugate base and 1 mol of H plus ion. | Gibbs Free Energy and Spontaneity.txt |
Which are also at a concentration of one molar. | Gibbs Free Energy and Spontaneity.txt |
Then we see the Delta G between the product and the reactants is given by 21.3 kilojoules or 21,300 Joules of energy. | Gibbs Free Energy and Spontaneity.txt |
Now, because the energy, the free energy of the products is higher than the free energy of the reactants, that means this reaction is andergonic. | Gibbs Free Energy and Spontaneity.txt |
And so we need to input 21.3 kilojoules of energy to actually drive this reaction in the forward direction. | Gibbs Free Energy and Spontaneity.txt |
So this reaction, as described here understanding conditions, is andergonic it's non spontaneous, it's reactant favorite. | Gibbs Free Energy and Spontaneity.txt |
And this is in accordance with the fact that formic acid is a weak acid and will not associate to a very large extent. | Gibbs Free Energy and Spontaneity.txt |
Now, just because the Delta G degree symbol the Delta G under standard state conditions is positive for these conditions, does not mean the Delta G will be positive under some other conditions. | Gibbs Free Energy and Spontaneity.txt |
In fact, by changing the Q value, by changing the concentrations of the reactants and products, we can ultimately transform this endergonic reaction into an exergonic reaction. | Gibbs Free Energy and Spontaneity.txt |
And this is a very important concept because it is continually used inside our body. | Gibbs Free Energy and Spontaneity.txt |
Our body changes the concentrations of energonic reactions to basically transform them into exergonic reactions. | Gibbs Free Energy and Spontaneity.txt |
Now, if we look at the following equation, this equation tells us exactly that. | Gibbs Free Energy and Spontaneity.txt |
So what the equation tells us is if this quantity is positive, then this doesn't necessarily have to be positive. | Gibbs Free Energy and Spontaneity.txt |
If this is positive, but this entire term is more negative than this is positive as a result of this Q value, then a positive quantity plus a negative value that is greater than this in magnitude will give us a Delta G that is negative. | Gibbs Free Energy and Spontaneity.txt |
And it's this Delta G, it's the sign of this Delta G, not this one, that ultimately dictates whether reaction is actually product favored or reactant favored. | Gibbs Free Energy and Spontaneity.txt |
And to see what we mean by that, let's carry out the following calculation. | Gibbs Free Energy and Spontaneity.txt |
So, in this particular case, we know that Delta G standard state condition is equal to 21.3 kilojoules. | Gibbs Free Energy and Spontaneity.txt |
Now, the question is what exactly should the Q value be for this Delta G to actually be negative and for our reaction to be spontaneous product favorite. | Gibbs Free Energy and Spontaneity.txt |
And the way that we're going to solve this problem is by basically using some type of negative value for this Delta G. So let's suppose Delta G is any negative value for, so let's suppose it's negative five kilojoules. | Gibbs Free Energy and Spontaneity.txt |
So this quantity is negative five kilojoules and this quantity is positive 21.3 kilojoules. | Gibbs Free Energy and Spontaneity.txt |