Thursday, November 12, 2009

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.
This leaves the proteins and Rubisco precipitated out in a pellet, defeated by the James Bond of this reaction, Ammonium Sulfate.
vs (rubisco)

So Ammonium Sulfate uses the property of..... SOLUBILITY!

Then we do dialysis where the pellet and supernatant are placed in DI water and the small particles flow out by diffusion....on to Part B.

Lab Two: SPECTROPHOTOMETRY

Spec-tro-pho-tom-etry...sound it out.

What's important in this lab?
  • What is the optimal range for the spec?
  • OD260=DNA absorbs light
  • OD280=Protein absorbs light
  • Why a Standard Curve is used?
  • OD=εcd
  • How to use a Spectrophotometer
  • Purity Ratio= OD260/OD280
KNOW WHAT THE OPTIMAL OD (absorbance) IS FOR DNA AND PROTEIN!!!
OD260=?
OD280=?

Optimal range for the Spec: 0.1-1.0

DNA Concentration= 50ug/mL x OD units x dilution factor
***if you dilute, you MUST multiply by the df to get the ORIGINAL solution concentration.

Think of it like this..
You pour a glass of milk.

It's white, opaque, and has 100% of its Calcium. Now, say you take that milk, pour half down the sink and fill it up with water (1:2 dilution).
Half your calcium went down the drain and was replaced with water! The glass only contains 50% of the Calcium you started with!

If your mom asked you how much Calcium was in the milk in the container (the un-diluted milk), you would have to multiply concentration of Calcium in the glass you have by 2 (df) to regain the Calcium you poured down the sink and find the ORIGINAL GLASS' CALCIUM CONCENTRATION!

Get it? Got it? Good!

Why do we construct a Standard Curve???
In Parts B, we used unknown samples.

How are we supposed to figure out their concentrations?
Plot the points on the Y axis--draw a line across and down to find concentration!

REMEMBER: OD is unit-less!

Serial Dilutions are used in this lab! Check the Dilution Blog to find more info!

Using the Spec is not what we are focusing on in this lab...what is it?
Find out before you do your lab write up! Use this as a guide:
  • What is the importance of OD in this lab?
  • What are we determining with the OD?
  • Why do we determine a purity ratio?


Lab One: SOLUTION MAKING!

I know what you thought when you started this lab. "Is this a joke? How dumb! I know how to dilute something, and why the hell are we making solutions the first day of lab? We are supposed to get out EARLY!!!"

Well I hope you didn't slack on those calculations because all those solutions will be used later this semester....in YOUR experiments!!!

So let's talk about dilution calculations...

Harder than it seems, right? But I'm going to break it down.
----------------------------------------

Volume to Volume Dilutions

This type of dilutions describes the ratio of the solute to the final volume of the diluted solution.

For example, to make a
1:10 dilution of a 1M NaCl solution, you would mix one "part" of the 1M solution with nine "parts" of solvent (probably water), for a total of ten "parts." Therefore, 1:10 dilution means 1 part + 9 parts of water (or other diluent).

Drops

For example: if you needed 10 mL of the 1:10 dilution, then you would mix 1mL of the 1M NaCl with 9mL of water.

Or: if you needed 100mL of the 1:10 dilution, then you would mix 10mL of the 1M NaCl with 90mL of water.

The final concentration of NaCl in both cases is 0.1M.

C1V1=C2V2

The Big Boy of Dilutions...know this!!!

Sometimes it is necessary to use one solution to make a specific amount of a more dilute solution. To do this, you can use the formula:

V1C1 = V2C2

where:
V
1 = volume of starting solution needed to make the new solution
C
1 = concentration of starting solution
V
2 = final volume of new solution
C
2 = final concentration of new solution

For example: Make 5mL of a 0.25M solution from 2.5mL of a 1M solution.

V1C1 = V2C2
(V
1)(1M) = (5mL)(0.25M)
V
1 = [(5mL)(0.25M)] / (1M)
V1 = 1.25mL

So you will need to use 1.25mL of the 1M solution. Since you want the diluted solution to have a final volume of 5mL, you will need to add ( V
1-V2 = 5mL - 1.25mL) 3.75mL of diluent.

Serial Dilutions

Sometimes it is necessary to dilute by a large factor so several serial dilutions are necessary.
To make a 1:10 dilution a 1:100 via serial dilution...

Take 1 part diluted to 9 parts water! This is another 1:10 dilution!

1:10 x 1:10= 1:100

Solutions made using Percentage by weight (w/v)

The number of grams in 100mL of solution is indicated by the percentage.
For example, a 1% solution has one gram of solid dissolved in 100mL of solvent. To make this type of solution properly, you should weight 1g and dissolve it in slightly less than 100mL. Once the solids have dissolved, you can bring the volume up to the final 100mL

Solutions made using Percentage by Volume (v/v)

In this case, the percentage indicates the volume of the full strength solution in 100mL of dilute solution.

To make a 60% Ethanol solution:

Step 1: Measure 60mL ethanol

Step 2: Add 40mL water to bring to 100mL

Molar Solutions

A 1 molar solution is a solution in which 1 mole of a compound is dissolved in a total volume of 1 litre.

For example:
The molecular weight of sodium chloride (NaCl) is 58.44, so one gram molecular weight (= 1 mole) is 58.44g. If you dissolve 58.44g of NaCl in a final volume of 1 litre, you have made a 1M NaCl solution.

To make a 0.1M NaCl solution, you could weigh 5.844g of NaCl and dissolve it in 1 litre of water; OR 0.5844g of NaCl in 100mL of water (see animation below); OR make a 1:10 dilution of a 1M sample.

Do it on your own:

How would you make a 500mL dilution of a 0.05M NaCl solution?

Answer will be posted when someone figures it out! Post your answers in the comment section!

Courtesy of http://www.wellesley.edu/Biology/Concepts/Html/volumetovolume.html

HOLD UP! How does this work??

So here's the deal...

Every lab will have its own blog. Questions, comments, interesting facts, and epiphanies can be posted in the "Comments" section.

How to ask a question:
  • Click the "comments" link
  • Type your question in "Comments" box
  • Choose a username to post as (AIM screen name, google account, etc.) in the drop box.
  • If you don't have one of these, create a google account. Go to google.com, click sign in. Below the sign in box there is a link "Create an account now", click and create an account. Use this to sign in, check out the blog, and post questions/comments.
  • Wait for an intern or another student to reply to your question. (Interns will check the student responses for accuracy)
  • Check back to see a response.
  • Still don't get it? Follow up with another question!


Easy as pie! Let's get started!

TAs and Professors are scary! Interns...Not so much.


Welcome to the BIOL3810 Molecular Cell Lab Intern Blog!

Hi, I'm Donna Burke.
Lab Intern Extraordinaire, Fall 2009!





Why I created this:

I, like most college-age students, have a fear of TAs and professors. I hate going to talk to them and I hate asking questions. They are smart, I don't want to waste their time with my questions that seem insignificant. This is why I created this blog, so students can talk to their peers, the interns, and get answers to the questions that seem small but are important to learning the material.

So here it is, the Molecular Cell Intern blog. This is for you, the students, to ask anything you want about Molecular Cell Lab! But as always, there are a few ground rules.....

Rules for the Blog:
  1. This blog is for BIOL3810 Molecular Cell Biology Lab ONLY!
  2. No homework answers, current lab reports, previous lab reports, past semester work, past semester exams, etc. will be tolerated. This is cheating and that's not cool.
  3. There will be NO whining about TAs, Professors, Interns, Classmates, Classwork, Lab Reports, or CTW. We can't change these...get over it.
  4. No ignorant or rude comments about questions or answers!! This is a happy place, a place for answers, this is not a place for mean spirits.
  5. Ask whatever you want, as long as it pertains to lab and the lab content!
Please remember, Interns are not gods of Molecular Lab. They did not write the lab protocols and are not experts in this field. They are here to help you understand the protocols, lab results, writing reports, and the basics of the lab. They are just undergrads, too!