Name: ______Date: ______Class: 1 2 3 4 5 6 7

The Case so far . . .

You have identified the strain of bacteria that infected the residents as ______.

You have identified the ink using ______and determined that the contaminated salad bag came from ______.

Gel Electrophoresis of Ink from SALAD Packaging

Testable Question: Can gel electrophoresis be used to confirm the paper chromatography results from the contaminated salad packaging?

Background Information: Agarose gel electrophoresis allows you to separate molecules according to size and electrical charge. What is agarose? It is a polymer that is made entirely of sugar molecules produced from seaweed and is used to give thicker consistency to foods such as ice cream. It is sort of like jello except that it does not get soft when it gets warm.

  1. What is agarose made from? Agarose is made from ______.

Molecules like ink dyes can move through an agarose gel, but just like in paper chromatography the larger they are, the slower they move. To understand the process, think about a backyardthat is full of trees. If you watch, you can see that small birds fly through the branches of the trees almost as if the trees were not there. What about a larger bird like a hawk or an owl? They have a harder time flying through the gaps. Therefore, they cannot fly as fast as the smaller birds. In the same way, small molecules can move quickly among the agarose branches in the gel, but larger molecules move more slowly because they have to find larger gaps to pass through.

  1. Why do smaller molecules travel further through the gel than larger molecules?

Smaller molecules move more quickly through the gel because ______

What is electrophoresis? Electrophoresis is a process that uses electricity to pull molecules from one place to another. If you look at the gel box, you will see that it has two bare wires called electrodes. One is connected to a red wire and has a positive charge, and the other is connected to a black wire and has a negative charge. Many, but not all, dyes have a negative charge so they are attracted by the positive charge and move through the gel towards the positive electrode. Therefore, we are going to use agarose gel electrophoresis to pull dye molecules through an agarose gel and separate them according to size and positive charge.

  1. What is electrophoresis?

Electrophoresis is the process that uses ______

  1. Which direction will negatively charged particles move?

Negatively charged particles are attracted to the ______and so they will move towards the ______.

  1. How is agarose gel electrophoresis used to separate mixtures of molecules?

Agarose gel electrophoresis is used to separate mixtures of molecules according to ______

Like the paper chromatography technique you have already used, this technique will allow you to separate the mixtures of the inks. You will compare the inkgel patterns used on the salad bagsof four salad processing plantsto the ink gel pattern on the bag that contains the contaminated salad. Then you willuse your data to conclude which bag (and therefore which processingplant) matches and is responsible for distributing the contaminated salad.

  1. How will agarose gel electrophoresis be used to confirm the results of the paper chromatography and prove which local farm produced the contaminated salad?

We will use gel electrophoresis to confirm the results from the paper chromatography lab by ______

Materials:

Name: ______Date: ______Class: 1 2 3 4 5 6 7

Salad Bag A Ink from Schneider’s Salads

Salad Bag B Ink from Kieffer’s Keep

Salad Bag C Ink from Endler’s Endive

Salad Bag D Ink from Godoy’s Greens

Contaminated Salad Packaging (x)

Agarose gel
TBE Buffer
Gel apparatus
Power supply
Pipettes and pipette

Gloves and goggles

Name: ______Date: ______Class: 1 2 3 4 5 6 7

Procedures (Check off each step as you complete it):

Safety Precautions

Follow your teacher’s directions in regardingmicropipette handling.

The power supply produces a voltagethat is high enough to cause severeelectrical shock if handled improperly.Ensure you know the procedures regarding the use of thisunit, making electrical connections, andthat you are directly supervised by theteacher.

Do NOT plug power supply into wallreceptacle until the safety cover ispositioned on the cell and all otherelectrical connections are properly made.

Make sure that all electrical equipment isdry.

Do not come in personal contact with orallow metal or any conductive material tocome in contact with reservoir buffer or theelectrophoretic cell while power supply ison.

The power supply may continue toproduce some voltage even when thepower has been turned off. Whendisconnecting the gel box be sure that theleads do not touch each other, come incontact with the buffer solution, orotherwise create any hazardous electricalcondition.

Loading and running the samples

____1.On the gel, load 10 uL of each ink sample into a corresponding lane. Keep track of which sample goes into which lane on the data sheet. Use a new tip for each sample and be careful not to puncture the bottom of the well. Your teacher will demonstrate.

____2.Clean up any spilled buffer or any other liquid surrounding the gel box thoroughly.

____3.Make sure the power supply is unplugged and switched off before proceeding.

____4.Carefully place the lid on the gel box (first making sure it is clean and dry) and connect the terminals correctly to the power supply.

____5.Have your teacher look over your connections before plugging in your power supply.

____6.Plug in the power supply, switch it on and adjust the voltage to 100 volts. You will know that current is circulating through your unit if you see tiny bubbles forming on the electrodes in the gel box.

____7.Let your gel run for 10 - 12 minutes. Keep checking your gel to make sure the ink molecules do not run off the end of the gel.

____8.When the fastest moving molecules are approximately 1 to 2 cm from the end of the gel, turn off the power supply. Disconnect the cords from the power supply and unplug the power supply.

____9.Ask your teacher to remove the gel tray from the gel box carefully – the gel is slippery and it can slide off and break. Place the tray on a flat surface and record your results on your data sheet. It will help if your gel tray is on a light background.

____10.Follow your teacher’s instructions for clean up. Micropipette tips may be discarded in the trash. Gel boxes and trays must be rinsed and air dried. Wash your hands thoroughly and clean the area where you were working.

STUDENT ACTIVITY Data and Analysis

1. Why is the comb placed in the center for this electrophoresis experiment?

The comb was used to create ______in the gel where you will place your samples of ink. The comb was placed in the center of the gel to allow ______

2. The diagram below represents your gel.

  1. As you load the ink samples into your gel, record which sample was placed into which well using the table below.
  2. After you have run the gel, use colored pencils or markers to draw in the results of your ink separation.

Black electrode (-)

Red electrode (+)

Lane Number / Factory Name/Source of Sample
A / Schneider’s Salad
B / Kieffer’s Keep
C / Endler’s Endive
D / Godoy’s Greens
E
F / Contaminated Package (X)

USE YOUR WARM UP FOR THE EXPLANATION HERE!

  1. Which ink (COLOR) mixture likely contained the smallest molecule? ______

Use evidence from your gel to explain how we know this.

______has the smallest molecule since the ______molecules will move______.

  1. Which ink (COLOR) mixture contained the largest molecule? ______

Use evidence from your gel to explain how we know this.

______has the largest molecule since the ______molecules will move______.

  1. List the inks (COLORS) that have molecules with a negative charge. ______Use evidence from your gel to explain how we know this.

______and ______have a negative charge since they both ______.

  1. List the ink (COLOR) that have molecules with a positive charge. ______Use evidence from your gel to explain how we know this.

______has a positive charge since it ______.

  1. Which salad bagmatches the contaminated salad bag sample? (FACTORY NAME AND LANE LETTER) ______Use evidence from your gel to explain how we know this.

______matches the contaminated salad bag because the ______and ______of the ink samples ______match.