Note to teachers This presentation is made available for use in your classroom. At miniPCR, we are committed to providing an engaging classroom environment for your students and provide this material to help you achieve that goal. Please feel free to modify this presentation for your needs as you feel necessary. Along with many of these slides are notes with added information and classroom tips. This presentation remains the property of miniPCR. Please do not redistribute. © 2017 by Amplyus, all rights reserved

blueGelTM Electrophoresis Rainbow Lab Release v4.1: October 2016 © 2017 by Amplyus, all rights reserved

Goals for today’s lab: Separate molecules based on size and charge-to-mass ratio using gel electrophoresis. Separate molecules based on polarity using paper chromatography. Learn to use a micropipette. Learn to use a gel electrophoresis system.

Gel electrophoresis is at the heart of DNA analysis

Gel electrophoresis is a method for separating molecules Gel electrophoresis separates molecules based on 2 main properties: size and charge-to-mass ratio. It is most often used with DNA or proteins, but can be used with any charged molecule. A scientist views protein separation results on a polyacrylamide gel

Today we will be using agarose gels Types of gels The two main substrates used in gel electrophoresis are agarose and polyacrylamide. Polyacrylamide gels are typically used for separating proteins. Polyacrylamide is made of long chains (polymers) of the molecule acrylamide, a known neurotoxin. Agarose gels are typically used for separating DNA. Agarose is a polysaccharide extracted from seaweed and is safe to handle. The material used to make the two types of gels is different, but how the gel works is similar. Today we will be using agarose gels

Gel Electrophoresis – agarose gels The gel is a matrix, similar to a web-like obstacle course, through which molecules are pulled by electric force. Smaller molecules have an easier time making it through the matrix, so they move faster. Larger molecules have more trouble making it through the obstacles, so they migrate slower. Molecules with a positive charge are pulled in one direction. Molecules with a negative charge are pulled the opposite way. The force is driven by an electric field, so a molecule’s charge determines how hard it is pulled. A molecule’s charge-to-mass ratio determines the speed at which it travels

Larger molecules move more slowly through the matrix. This illustration shows a simplified model of molecules being pulled through the matrix. It can also be useful to show students a video clip of the game “Plinko” on the game show the Price is Right. The game show “The Wall” also has a similar board. When showing the clip, ask students, “How fast would the chip/ball fall if it were the size of a marble? A baseball? A marble?”

Molecules will move towards the opposite charge + - + - In this animation, note to students that the molecules will be pulled towards the charges at either end of the matrix. Students may be confused as to why the molecules are not attracted to each other. In actuality they will be; however the electric field placed on the matrix by the electrodes is much, much more powerful.

The larger the charge-to-mass ratio, the faster the molecule will move through the gel 2- In this illustration note that all molecules have a negative charge and so are pulled in the same direction. You may note that when working with DNA, all DNA molecules have the same charge to mass ratio and so only size is a factor in separating molecules. Charge-to-mass ratio will be an important factor in determining how quickly dyes move in the gel. - + 3- (molecules of equal mass)

Paper chromatography separates molecules based on solubility in a solvent Polar molecules are those whose charge is not spread evenly over the molecule. While the molecule as a whole may have no charge, one part of it may be more positive and another part may be more negative. Water and ethanol (rubbing alcohol) are polar solvents. Molecules that very polar will dissolve easily in them. Molecules that are not very polar will not dissolve as easily. Water Ethanol Paper chromatography is one of may types of chromatography. Chromatography is any procedure that separates molecules by passing them through a medium in which the molecules move at different rates. Paper chromatography can be useful because it is extremely easy and inexpensive to run. In both the water molecule and the ethanol molecule, the red ball represents an oxygen atom; the white balls represent hydrogen atoms. In a bond between oxygen and hydrogen, the oxygen tends to be more negatively charged, while the hydrogen atom tends to be more positively charged. In the case of ethanol, the black balls represent carbon. There is no polarity on the portion of the molecule containing carbon.

Polar molecules will travel farther in paper chromatography In paper chromatography, the solvent will travel up the filter paper by capillary action. Molecules that are very well dissolved will move easily with the solvent and travel far up the paper. Molecules that are not as well dissolved will not move as easily with the solvent and will not travel as far up the paper.

Today’s Lab To become familiar with the gel electrophoresis system, today we will use gel electrophoresis to separate food dyes. You will predict which molecules will travel the fastest through the gel and in which direction each molecule will travel. You will compare your results to another separation technique, paper chromatography.

Like everything, the food you eat is a collection of different molecules. Many foods you eat are the color they are because dyes have been added. Dyes are small chemical molecules that are safe. The dyes used in this lab are commonly used food dye molecules.

Dyes are small molecules that have different sizes and chemical charges Orange G https://en.wikipedia.org/wiki/Allura_Red_AC Allura Red AC Methylene Blue Pictured are three of the molecules that will be used in todays lab. Students can easily recognize that Allura red is the largest, which should cause it to move more slowly through a gel. In solution the Na and Cl atoms will separate from the rest of the dye. The Allura Red will be left with two negative charges noted on the oxygen molecules. The Orange G will also have two negative charges at the oxygen atoms. The Methylene blue will have one positive charge at the sulfur atom.

Let’s do it! Micropipetting Gel electrophoresis Dye visualization While the gel is running students will run the paper chromatography. 45 min total

Warming up: Micropipetting practice

Micropipetting practice Steps to micropipetting Identify volume range Adjust volume Press plunger to FIRST STOP Slowly Release Plunger Now press plunger to SECOND STOP Practicing pipetting is optional but can be extremely useful for students new to using pipettes. Today students will use 20 microliter pipettes. These pipettes usually have three numbers on the dial. The first number represents the tens place, the second number represents the ones place and the third digit represents the tenths place.

Now, transfer real liquids: 20, 10, and 5µl Adjust volume Get a tip Press plunger to FIRST STOP Slowly release plunger, collecting liquid Transfer liquid Press plunger to SECOND STOP Remove tip from liquid Release plunger Eject tip The biggest difficulty ties students usually have when new to pipettes is reading the volume properly and recognizing the difference between the first and second stop. Students can practice by pipetting water between tubes or onto strips of parafilm. Adding som food coloring to the liquid can make it a little easier for beginners.

blueGel™: integrated electrophoresis AND visualization system Cast 0.8% agarose gels (instructions are per gel): In an Erlenmeyer Flask add: 25 ml 1X TBE buffer 0.2 g agarose Microwave for 30 seconds or until liquid is clear. Cool for 1-2 minutes before pouring. Allow gel to cool to harden (about 15 minutes) Pouring gels can be done by students or in advance by the teacher. If done by students you may need to take more than one 45 minute class period. Gels can be poured in advance if kept if TBE buffer. Remember to place the comb in the middle of the casting tray for this lab. When cool, place the gel and casting tray in the blueGel electrophoresis system. Cover with 1X TBE buffer

The wells in our gel are in the middle of the gel Usually in an agarose gel, the wells are cast at one end of the gel and that end is placed near the negative electrode This is because DNA is negatively charged and will run through the gel towards the positive electrode. The dyes in this lab may be positively or negatively charged and so may run in either direction

1. Draw up 15 µl of dye using a micropipette 2. Carefully load each dye into the correct well of the gel. Follow the Use a new tip each time to avoid contamination

Load gel as follows: Don’t pierce bottom! 1 2 3 4 5 6 7 Bromocresol Purple Allura Red AC Unknown A Unknown B Bromophenol Blue Methylene Blue Orange G 1 2 3 4 5 6 7 A common mistake for people new to loading gels is to pierce the gel with the pipette tip. The tip needs to just barely enter the top of the well. Because the dye is heavier than the buffer, the dye will sink to the bottom of the well. Lining up the tip with the well can be difficult for new users. As in the picture on the previous slide, it can be easier to hold the pipette at an angle and approach the well from the side, in the direction that the wells are the longest. It can also be helpful to rest the tip end of the pipette on a finger from the hand not holding the pipette. This can steady the pipette making lining it up with the well easier.

Prediction (direction, distance) Predict results Which dyes will travel the farthest? Which dyes will travel which direction? Dye Color Molecular Weight Charge Prediction (direction, distance) Bromophenol Blue Purple 669.96 g/mol - Methylene Blue Blue 373.90 g/mol + Orange G Yellow 452.38 g/mol Bromocresol Purple 540.22 g/mol Allura Red AC Red 496.42 g/mol H Dyes with a negative charge should run toward the positive electrode. Dyes with a positive charge will run toward the negative electrode. Larger dyes should not run as far as smaller dyes. The difference in charge to mass ratio and the shapes if the molecules will account for deviations from this pattern.

Tweet out your results! @miniPCR #RainbowLab Wrap up How did the experiment turn out? Which dyes ran in each direction? Can you explain to me why? Which dye traveled the farthest? Which dye traveled the least distance? Can you explain why? Compare gel electrophoresis and paper chromatography? What are some ways that paper chromatography and gel electrophoresis are similar? In what ways are they different from each other? Can you think of a way that molecules may travel together in one technique, but separate in the other? Tweet out your results! @miniPCR #RainbowLab

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Appendix: additional resources Understanding gel electrophoresis http://www.yourgenome.org/facts/what-is-gel-electrophoresis https://www.khanacademy.org/science/biology/biotech-dna- technology/dna-sequencing-pcr-electrophoresis/a/gel-electrophoresis Understanding chromatography https://www.khanacademy.org/test-prep/mcat/chemical- processes/separations-purifications/a/principles-of-chromatography http://www.chemguide.co.uk/analysis/chromatogrmenu.html History of the use of DNA in crime solving http://www.forensicmag.com/articles/2005/01/evolution-dna-evidence- crime-solving-judicial-and-legislative-history

The complete biotech toolkit: DNA Discovery System™ Micropipetting PCR Gel electrophoresis Visualization