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Measurement (Team 5)

Page history last edited by Emily Wasik 10 years, 6 months ago

A. Learning Objectives 

In this lab, students will:

•  explain why scientists use gel electrophoresis.

•  measure volume and weight in units of liters and grams.

•  become familiar with the use of the micropipettor.

•  learn about the process of gel electrophoresis

 

B. Textbook Correlation: 

Please review Section 20.1 Gel Electrophoresis Can Separate Macromolecules, Such as DNA when preparing for lab.

 

C.  Introduction to Gel Electrophoresis

In your introduction for your presentation explain why scientists use this technique.  Also discuss the technique itself, including the function of agarose, running buffer like Tris-acetate-EDTA (TAE), DNA loading dye, and an electrical current.  Describe what two properties of nucleic acids/proteins are measured using this technique.  Include an image/animation when appropriate. 

 

     Gel electrophoresis is a technique that is used to separate the different macromolecules, these being deoxyribonucleic acid (DNA) and proteins. Scientists use this technique in order to assess the results of a specific cloning experiment. Gel electrophoresis allows scientists to determine and evaluate the lengths and size of the DNA fragments that have been inserted into the recombinant vectors. DNA fragments are separated according to their sizes as they run through the gel; this helps scientists to determine whether one type of DNA is identical to another type of DNA or not (for example in paternity tests or criminal cases). The two properties of nucleic acids/proteins that are measured using this technique are the charge and size of the nucleic acids/proteins.

     Gel electrophoresis involves the use of an electrical current that runs through the gel in order to separate the macromolecules, DNA and proteins. The negatively charged DNA and proteins are attracted to the current coming from the opposite end of the wells. The current "drags" the molecules through the gel. The movement of the DNA and proteins will be determined by two major factors: the charge and the relative size of the macromolecule. 

     First, the samples of the DNA or protein are loaded into wells that are located at the top of the gel. These wells are depressions that are found at the surface of the gel. Each sample is loaded into its own well so there is the possibility of multiple samples being tested at the same time without any fear of contamination. The gel that is used during this process is called an agarose gel which is a flat semisolid gel. Agarose gel is a polysaccharide matrix that allows for the DNA and proteins to run through it. Its major function is to act as a sieve in order to catch the macromolecules as they are carried by an electrical current through the gel. This gel is submerged in an electrophoresis buffer that contains charged ions and maintain the pH. One of these running buffers, Tris-acetate-EDTA (TAE), is loaded into the well along with the sample. This buffer helps to keep the sample in the well and to track the location of the sample when it travels through the gel. Loading dyes are also placed into the well with the sample and the running buffer. These dyes allow the DNA to become more visible in the gel and helps scientists to easily record data on the lengths that were traveled by the sample as well as its base pair and the number of bands that are present in the gel itself. Molecules that are lighter will be able to travel through the thick gel easier than heavier molecules, thus the lighter molecules will be able to travel farther through the gel.

     After all the samples are loaded with their appropriate buffers and dyes, an electrical current is then applied to the gel. This electrical current is a very important factor in gel electrophoresis. One end of the gel is positively charged while the other end is negatively charged. Molecules will migrate towards the side of the gel that is opposite to their charge. For example, DNA is a negatively charged molecule so it will migrate towards the positively charged end of the agrose gel. This electrical current that exsists in the gel helps scientists to determine the relative charge of the given molecule.

 

 

D.  The Process of Electrophoresis

This video is a short introduction to preparing an agarose gel.  PLEASE VIEW BEFORE COMING TO LAB:


 


Step 1 - Preparing the Agarose Gel:  
The final agarose concentration used in our experiment today is 1%.  Solution percentages are a function of mass of substance/total volume of solution.  To make 100mL of a 50% solution, you would dilute 50g of solute in 100mL of solvent.  You can think about these solutions in terms of g/mL.  In the first step today, we will make 25mL of a 1% agarose solution.

 

Question:  How many grams of agarose are required in this dilution?  Show your math.

 

mass/total volume= percent of dilution

 

x g/25 ml = 1%

x g/25 ml = 0.01

1x= 25(0.01) ml  

1x = 0.25 g    

x = 0.25 g          

The amount of agarose required in this dilution is  0.25g. 

 

(In the experiment we performed during class, we changed the amount of agarose to 0.35g as per request of Dr. Weber). 

 

 

 

How to Measure Mass

Mass is the amount of material in an object. Mass stays the same no matter what force (i.e. gravity) is acting on the object.  To measure mass, the metric unit is the gram, which is the approximate weight of a small paper clip.  A balance is the most common instrument used to measure weight or mass; there are many different types of balances.   In the lab, we use a digital balance that is accurate to the second decimal place (1.00g) (insert image of balance)  weigh an object using the digital balance:

1.  Turn the balance on.  Wait until the read-out shows 0.00g.

2.  Center a weigh container on the balance and press the ZERO button.  The weight of the container will be subtracted out of the final measurement, allowing only the object of interest to be weighed.

3.  Place the object of interest in the center of the weigh container and record/note the weight.

 

Step 2 - Preparing the Running Buffer:  It is common to keep solutions in the lab at higher concentrations than used in experiments.  These solutions are called stock solutions.  The rationale behind this practice is to limit the amount of solution that must be stored.  For example, a stock solution may be used at a final concentration of 5x, but stored as a 100x stock solution.  In order to prepare the 5x solution from its stock, a 1:20 dilution is required.  You can figure out how much to dilute using the simple equation M1V1 = M2V2.  In this scenario, if I need 100mL of a 5x solution:

 

Equation: (5x)(100mL) = (100x)(mL)

 

Solve for V2: 500/100 = V2

 

Answer:  I need 5mL of 100x stock diluted to 100mL total.

 

A 1x concentration of TAE is used as the running buffer.  This solution is not only used to run our gel, it is also used to dilute the agarose.  The stock solution of TAE in the lab is 50x.   In the space provided below, calculate the volume of 50x TAE required to produce 400mL of 1x TAE.  SHOW YOUR MATH.

 

Work Shown Below: 

 

To find the volume of 50x TAE:                                            To find the volume of dH2O:

 

M1V1 = M2V2                                                                        400mL of total solution - 8 mL of 50x TAE = 392 mL of dH20                                               

(1x)(400ml) = (50x)(ml)                                                      

400 = 50V2                                                                                                                                                         

V2 = 400/50                                                                     

V2 = 8 ml                                                                         

volume of 50x TAE
   8 mL  
volume of dH2O
   392 mL      
total volume 400 mL 

 

 

 

General Tips Regarding Volume Measurements: 

Volume is the amount of space a substance occupies. The liter is the basic unit of volume. Various devices are used to measure the volume of liquids depending on the volume being measured and the accuracy required. For larger volumes, biologists use glass and plastic measuring vessels, such as graduated cylinders and volumetric flasks. Beakers and Ehrlenmeyer flasks often have lines indicating volumes, but these lines are approximate; therefore, never use a beaker or flask as a measuring device. Pipettes are usually preferred for volumes in the 1-25 mL range. Various micropipetting devices are commonly used to measure volumes in the microliter range.

 

Once the solution is made, the agarose must be heated to drive the powder into solution.  The best practice is to set the timer for a minute and microwave on high until you see the solution begin to boil.  Remove from microwave oven and swirl and check to see that all the agarose has dissolved. Your solution should be clear.  Allow the solution to cool to 55-60ºC or until you can place your hand comfortably on the flask for a few seconds.  If your solution begins to solidify before you have a chance to pour it, reheat the solution in the microwave.

 

Step 2 - Setting up the Electrophoresis Casting Tray:   Prepare the casting tray by using the metallic gel casting gates provided.  Be careful that the tray is sealed well and in the proper orientation; otherwise, the agarose solution will leak onto your work surface before it has a chance to solidify. 


Step 3 - Pour the Gel:  Place the comb in the grooves of the casting tray in the middle of the tray.  Pour the agarose solution into the tray to where it comes a little over halfway up the comb.  Allow the gel to solidify at room temperature.

 

Step 4 - Add the 1x TAE to the Running Chamber:  Remove the casting gates and add 300mL of 1x TAE buffer to the running chamber.  Once filled, carefully remove the comb.  The wells should fill with buffer.

 

Step 5 - Preparing your samples:  When working with molecules like nucleic acids and proteins, scientists often work in units of microliters (1/1,000,000 or 10-6 liters).  An instrument called a micropipettor is utilized to accurately measure volumes in units of microliters.  Proper use of this instrument will ensure bith precise and accurate measurement.  Here is a short video on proper use of the micropipettor.  PLEASE VIEW BEFORE COMING TO LAB:

 

Proper Use of a Micropipettor

Micropipettors are among the most common instruments used by molecular biologists.  It is essential that they be used properly, otherwise, any work done in a molecular biology laboratory is suspect. Richard Curtis and George Rodrigues published a study in which they evaluated the micropipetting accuracy and precision of students (with experience pipetting) and experienced technicians (Pipet Performance Verification: An Important Part of Method Validation, Curtis and Rodrigues, American Laboratory, Feb. 2004, 12-17.) The results of their study were alarming: many students and technicians pipetted incorrectly. Some of the individuals tested has per cent inaccuracy scores as high as 35% (or more). The imprecision, reported as coefficient of variation, was as high as 68% and almost always exceeded the specifications for the pipettes, as given by the manufacturers. In other words, many of the people tested had very poor pipetting skills. It is important not to take pipetting for granted. We will simulate a very common molecular biology technique, PCR, which requires good pipette skills.  It is also important to remember that micropipettors are expensive and fragile. ALWAYS USE PROPER TECHNIQUE TO AVOID DAMAGE TO THEM. Here are some tips to avoid damaging the instrument:

  • Never drop a micropipettor.
  • Never rotate the volume adjuster of an adjustable micropipettor beyond the upper or lower range of the instrument.
  • Never pass a micropipettor through a flame.
  • Never use a micropipettor without a tip thus allowing liquid to contaminate the shaft assembly.
  • Never lay a filled micropipettor on its side thus allowing liquid to contaminate the shaft assembly.
  • Never immerse the barrel of a micropipettor in liquid, only immerse the tip.
  • Never allow the plunger to snap up when liquid is being aspirated.
  • Store micropipettors set to their highest volume. This releases pressure on the spring inside the micropipettor.

 

 

 Today we are laoding dyes of various sizes and charge (xylene cyanol, bromophenol blue, orange G, safranin O, a mix of all four, loading dye).  Adding dyes to a DNA sample allows one to assess how "fast" your gel is running.  They also  render your samples denser than the running buffer (so that the samples sink in the well).  The first four dyes come as a 1x stock solution.  Please dilute each to a 0.25x solution in a final volume of 20 microliters.   Loading dye comes prepared as a 5x stock solution; dilute the loading dye to a 1x final concentration in 20 microliters.  Show your math below, including the equation you used to calculate:

 

Work Shown Below:

 

Volume of 1x Dye

(Final X)(Final Volume)=(Stock X)(Variable)

(.25x)(20 microliters)=(1x)(microliters)

5/1 microliters

5 microliters of 1x Dye

 

Volume of dH20 (for 1x dye)

(Total Volume) - (Volume of 1x Dye) = (Volume of dH20)

20 microliters - 5 microliters = 15 microliters

15 microliters of dH20 for 1x dye

 

Volume of 5x Dye

(Final X)(Final Volume)=(StockX)(Variable)

(1x)(20 microliters)=(5x)(microliters)

20 microliters/5 microliters= 4 microliters

4 microliters of 5x Dye

 

Volume of dH20 (for 5x dye)

(Total Volume) - (Volume of 1x Dye) = (Volume of dH20)

20 microliters - 4 microliters = 16 microliters

16 microliters of dH20 for 5x dye

 

 

 

 

volume of 1x dye (xylene cyanol, bromophenol blue, orange G, safranin) 5 microliters  volume of 5x loading dye
  4 microliters
volume of dH2O
15 microliters  volume of dH2O
          
16 microliters
total volume 20 microliters
total volume 20 microliters

 

Step 6 - Loading your samples:  We will load six samples onto the gel with each member of the group responsible for three.  Please use the center four lanes and keep the outermost lanes free of sample.  Here is a helpful video of the process.  PLEASE VIEW BEFORE COMING TO LAB.


 

 

Step 7 - Running the Gel:   Once all the samples have been loaded into the wells, place the cover over the electrophoresis chamber.  Is your gel oriented in the right direction?  Attach the electrodes to the electrophoresis chamber and then to the power supply.  Place the cover over the electrophoresis apparatus.  Turn on the power supply.  Do not stick your fingers or anything else into the buffer.  You will get electrocuted!  Run the gel at 50-100 volts for approximately 30-40 minutes.  When the loading dye front has run 3/4 the length of the gel, turn off the power supply, unplug all the electrodes, and, with gloved hands, remove the gel form from the electrophoresis to visualize the gel.  Make sure the orange dye front is still visible on the gel.

 

Step 8 - Measuring the Migration Distance:  The meter is the common unit of length in the metric system.  In this part of the exercise, we will measure the migration of the three dye fronts using units of millimeters  (1/,1000 or 10-3m).   There are two dyes in the loading dye solution you used today (Xylene cyanol and bromophenol blue).  This table obtained from openwetware.com (retrieved 31 August 2010) draws a relationship between the migration of the dye and the the migration of DNA fragments.  In the image on the right, you can directly compare the three dyes fronts and actual DNA fragements.  The benefit of including the dyes is that DNA cannot be observed with the naked eye.  Therefore, one can estimate where their DNA fragment of interest is on the gel based on the position of each dye.

 

 

First document the direction of movement (towards the negative or positive electrode).  Measure the distance traveled for each dye front in mm.  Next, calculate the rate of movement trough the gel in mm/min.

 

Dye
direction of migration  distance migrated (mm)
rate of migration (mm/min)
Xylene cyanol Towards positive  10mm  1mm/min 
Bromophenol blue Towards positive  14mm  1.4mm/min 
Orange G Towards positive   17mm  1.7mm/min 
Safranin O Towards negative  12mm  1.2mm/min
       

 

Questions:

1.  Which dye molecule has the greatest molecular weight?  Which has the lowest molecular weight?  Explain your reasoning.

Orange G dye has the greatest molecular weight. Xylene Cyanol has the lowest molecular weight. The larger the molecule, the slower the rate at which it moves through the agarose gel. Thus, since Xylene Cyanol moved at the fastest rate of migration, it was the smallest molecule. Similarly, since Orange G moved at the slowest rate of migration, it was the largest molecule.  

 

2.  Which dye molecules were negatively charged?  Which were positively charged?  Explain your reasoning.

Xylene Cyanol,  Bromophenol blue, and Orange G were all negatively charged dyes. We know that opposite charges attract and since the three dyes mentioned moved towards the positive electrode, we can conclude that they contain a negative charge. On the other hand, we can also conclude that Safranin O is positively charged because this dye migrated toward the negative electrode.   

 

3.  Imagine you have a 500bp DNA fragment you want to isolate.  Calculate the rate of migration for this fragment.  What if you were to run the gel for 180 minutes?  Would you still be able to isolate the fragment on the gel? Explain. 

 

 

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