BIOL3810 Molecular Cell Intern Blog

Lab Seven: Polymerase Chain Reaction

PCR! Who done it??

As a kid, I loved mystery books! Loved them! So when this lab came around, I was excited!

Mystery in the Molecular Cell Lab!

I think it was Dr. Cruz... in the lab... with the graduated cylinder!

What's important in this lab?

What is PCR?
What is Taq Polymerase?
What are the components of PCR?
What are the Steps of PCR? Where do they take place?
Primers are...
What is a genetic repeat? Polymorphism? Short Tandem Repeats?
Why do we want so many copies?

PCR

-An alternative to cloning. It amplifies specific gene sequences by using Taq Polymerase and sequence specific primers.

Taq Polymerase was isolated from Thermus aquaticus and is thermostable (not denatured by heat)! So why do we use Taq?

PCR Components include:

DNA (genomic or cDNA)
Taq Polymerase
SEQUENCE SPECIFIC Forward and Reverse Primers
Deoxynucleotide Triphosphates (dNTPs)
Reaction Buffer

STEPS!!!

-Denaturation: 95 degrees! HOT HOT HOT!

-peels double stranded DNA into single stranded DNA for copying!

-Annealing: 55-65 degrees!

-SEQUENCE SPECIFIC forward and reverse primers bind to ssDNA on sequence before target sequence

-Elongation: 72 degrees!

-Taq binds to primer and elongates the strand

REPEAT 20-30 times!

Make BILLIONS of copies!

Check out this youtube video! Super Boring, but does the trick!

Know what a Polymorphism is! A Short Tandem Repeat!

Don't get it? Post Questions!

What do our gels tell us?

Compare the Crime Scene DNA bands to Suspects 1-4 bands. Any near matches?

Any this is just for fun....I suggest watching it!

Biologists have too much free time while waiting for the thermocycler....

Lab Six: Enzyme Kinetics!

We like it fast, give us some enzymes!

The last step of Glycolysis:

PEP + ADP --> Pyruvate + ATP

Enzyme: Pyruvate Kinase

What's important in this lab?

What is the importance of an enzyme?
What is Km?
What is Vmax?
What does DNPH do?
What does NaOH do?
Why do we add the Pyruvate Kinase last?
What does a Michaelis-Menton Plot show?
What does a Lineweaver-Burk Plot show?
Why is time so important?
What is an activator? an inhibitor? How can you tell?

Enzymes:

The energizer bunny of reactions!

It keeps going and going until there's no substrate left! They lower the activation energy of the reaction so it takes less to get to the final product!

Km-the michaelis constant

This is the measure of the affinity of the enzyme for the substrate.

Km= 1/2Vmax

1/2 Vmax? What's Vmax?

The maximum rate an enzyme can convert a substrate into a product.

There's only so fast the bunny can pound that drum before his arms fall off! That is his Vmax!

What does DNPH do?

Gives the Pyruvate color so as to absorb light during spec at 510nm.

What does NaOH do?

NaOH is a used to alter pH. It alters the solution's pH and stops the

reaction! IMPORTANT! If the reactions are not timed correctly and runs long, more product is made in one reaction than another!!!

We add Pyruvate Kinase last so the reaction starts when the enzyme is added!

Michaelis-Menton Plot

Velocity vs [Substrate]

What is your substrate?

Lineweaver-Burk Plot

1/V vs 1/[S]

This gives a more accurate Vmax (crosses at y-axis) and [Substrate] (crosses at x-axis)

Activators and Inhibitors

What is an activator?

Something that promotes a reaction!

What is an inhibitor?

Something that slows a reaction! Feedback inhibition plays a large role in Enzyme Kinetics!

Let's have story time:

I love to drive! Love it! But this means I have to fill up with gas, ugh! No matter what time of year, I hate standing and holding the gas pump handle!!! So I always put the clip on and walk away. Now why am I telling you about my love for driving and my hate for filling up??

Feedback inhibition is like the automatic click off of the gas pump! The pump is the enzyme, the tank is empty and wants to be filled with our product, gas! The pump continues to produce gas, our product, until the tank reaches capacity and tells the pump to STOP! If the pump did not stop the car would overflow with gas all over the station. The feedback from the tank saying it is full is the same as the product in our Enzyme Kinetics reaction, it tells the enzyme to STOP!

Lab Five: Subcellular Fractionation

What's important in this lab?

What subcellular components are being separated?
Why do we use centrifugation?
What are the expected results?
What are in the specific fractions?
What does Succinate Dehydrogenase do?
Why do we use OD260?

In this lab, we are isolating mitochondria and nuclei from liver cells by centrifugation.

Centrifugation separates subcellular particles by density, nuclei are more dense than mitochondria.

If nuclei are more dense, do they pellet out of solution in the low speed or high speed spin?
Where do we find them after spin one? Pellet or supernatant?

-It takes LESS speed to pellet out the nuclei because they are MORE dense!

-It takes MORE speed to pellet out mitchondria because they are LESS dense!

Why are nuclei more dense?

Because there is more DNA!

What are the expected Results?

If the more dense particles pellet first, then...

-the nuclei are in P2K

-the mitochondria are in S2K

If the less dense particles pellet second, then...

-the mitochondria are in P20K

-the tiny particles are in S20K

SDH Assay

Succinate + FAD --> Fumarate + FADH2

***The reduction of FAD to FADH2 creates the color change!!!***

What does color change in SDH assay tell us is present?

-mitochondria

-if mitochondria are present, a Redox reaction is taking place and causing the blue to colorless change

So, if the expected results show that S2K SHOULD contain the mitochondria,

what color will it be?

Colorless!

But what if P2K is colorless? What does that mean? How could we fix it?

-Color change tells us that mitochondria are present! Uh oh!

-If we spun the mitochondria out in the first spin, the spin is too fast! Slow it down and try again and see if the mitochondria stay in the supernatant!

****THIS IS HUGE! Know what is present, what it means, and how you fix it!

Determining Concentration of DNA

Spec at OD260 (Optimal light absorption for DNA)

[DNA]= OD260 x 50ug/mL x df

If you cant remember dilution calculations, check out the Spec lab and the milk story!

Lab Four: Restriction Digest

pAMP, BamHI, EcoRI, and HindIII are the main players in this game!

What is important in this lab?

What do restriction enzymes do?
What are sticky ends? Blunt ends?
What are plasmids? pAMP?
Why do we leave some restriction enzymes out of the mixes?
Why do we use gel electrophoresis?

What do the enzymes do?

They cut at specific sequences. They make circular plasmids into linear strands of DNA!

What happens when you put in more than one enzyme?

You get multiple cuts which means multiple strands of DNA.

Why do we use gel electrophoresis?

To determine the size of the fragments after the cuts
To determine the plasmid map and where in relation to one another the enzymes cut.

Lab Three: ION EXCHANGE CHROMATOGRAPHY Part C

Part C: Electrophoresis

What's important in this lab?

What property is electrophoresis using?
How will we determine if Rubisco is present? Can we?
What are the molecular weights of the 2 Rubisco polypeptide subunits?
What travels fastest/farthest? Small proteins or large proteins?

So we've used solubility and charge to isolate Rubisco from Spinach leaves. What's left?

SDS Page uses a gel to run the proteins (Filtrate, P1, P1(low/med/high), P2, P2(low/med/high), and marker).

Why is the marker important?

We know the molecular weights of the 2 Rubisco sub-units (55,000 and 14,000). The marker will allow us to know the molecular weights of the proteins in the pellets and supernatants.

Did you find Rubisco?? Compare your results to the marker! (Notice, other proteins are still in the mix, see why we did all these steps?!)

Smaller proteins travel faster and farther! They end up at the bottom of the gel! Large proteins are too fat to move, they move sloooowww!

Here's a way to remember this: (compliments of Dr. Poole)

I'm a girl, and if I do say so myself I am a thin girl! My friends on the other hand, are 6ft 5in, 250lb men! HUGE! When we go out, they can see over the crowd, but getting to the bar is another story, they are toooo big!

Not going to lie, I want a drink when I'm there and I'm not willing to wait! So my little self can weave in and out of the crowd and make my way to the bar! DRINKS FOR ALL! I win because I am small, I can get through the crowd faster than my 250lb friends, all I need them to do is grab their drinks over everyone else's head!

So technically, I am the protein in the gel. I am smaller so I can get to the finish faster, I get to the end (the bar) before everyone else!

Lab Three: ION EXCHANGE CHROMATOGRAPHY Part B

Part B: Ion Exchange Chromatography

So in Part A, we isolated the proteins, including rubisco, using Solubility.

So in Part B, what are we doing? Isn't that enough?

No...we want RUBISCO not the other mess!

What is important in this lab?

What property is being used to separate the proteins?
What charge do the beads carry?
What charge is Rubisco?
What is the purpose of the low-salt buffer? medium-salt buffer? high-salt buffer?
Why do we collect all the flow through?
Which flow through contains Rubisco?
Why do we take the OD of all the samples?

Big Players at the Table:

Low Salt Buffer, Medium Salt Buffer, High Salt Buffer, and Proteins.

So through your research, you should have determined Rubisco has an overall NEGATIVE charge!

So we want to use a POSTIVELY CHARGED COLUMN! So what are we using to separate the proteins?? CHARGE!

Let's talk about the Ion Exchange Column...

The resin contains positively charged cellulose beads that hold onto the negatively charged proteins.

At this point, it's a battle of the strengths. Salt Concentration strengths, that is.

So we put in the supernatant and pellet (into different columns of course). Then we use the salt buffers to battle with the proteins.

Low Salt:

Competes with the very low charged proteins that have bound to the column. Those that have a very low negative charge will come out in the flow through of the low salt buffer.

Medium Salt:

Competes with the medium charged proteins on the columns. This knocks the medium negatively charged proteins into the flow through.

High Salt:

Competes with the high charged proteins on the columns. This knocks the strong negatively charged proteins into the flow through.

So where do we expect the Rubisco?

Pellet or Supernatant?
Low Salt?
Medium Salt?
High Salt?
What does the OD reading tell you about the amount of protein in the flow through? Is it rubisco? Can we tell?

There's another part...keep on truckin'!

Lab Three: ION EXCHANGE CHROMATOGRAPHY

MMmmm..Spinach!

Part A: Ammonium Sulfate Precipitation

What's Important in this Lab?

What are we isolating?
What is Rubisco?
Is its overall charge Positive or Negative?
What are the 3 properties used to isolate Rubisco?
What property does Ammonium Sulfate Precipitation use?

Rubisco, the most abundant protein in plants.

So why do we use OD280 in this lab??

I hope you learned in the Spec lab that the optimal OD for proteins is 280!! Convenient, eh?

So what's the deal with Ammonium Sulfate Precipitation? What in the world is going on???

Let's imagine a love triangle, starring:

Water as Megan Fox

Ammonium Sulfate as Daniel Craig

Rubisco (and other proteins) as Shia Le Beouf

In the beginning, Megan and Shia are in love, attached at the hip by hydrogen bonds.

But along comes Daniel Craig, so big, so buff, and has a way hotter charge!

Megan (water) comes in contact with Daniel (Amm. Sulf). Shia (Rubisco/proteins) unfortunately needed to hit the gym because Daniel's strong charge pulls Megan to him and sends Shia to precipitation.