In this laboratory exercise you will participate in two different activities. You will extract and spool DNA from liver cells and you will make a DNA fingerprint from pre-prepared samples. You will be using equipment and chemicals that are not used in other labs in this course and require special precautions. Read the following instructions and warnings carefully. Email your instructor with any questions you may have regarding the use of the equipment or chemicals before class or speak to them directly before you proceede.
| Goals | Gel Electrophoresis Apparatus |
| Safety | Other Materials |
| Micropipettor | Tips for Success |
The goal of this lab is to give students an opportunity to isolate DNA, complete a restriction enzyme digestion, and to load, run and analyze a gel.
General:
DNA fingerprinting requires the use of a gel electrophoresis apparatus (the apparatus and its components will be discussed more thoroughly below).
To visualize DNA in the completed gel you must use either Carolina Blue or ethidium bromide. Carolina is non-toxic. Ethidium bromide is a known carcinogen and should be handled VERY carefully. Your instructor should stain your gels, if your are enrolled in a flexible learning section read the safety instructions below carefully.
Equipment/Procedures Review
In this lab you will use a horizontal electrophoresis apparatus to separate DNA fragments and a micropipettor to dispense your samples into the gel wells.
Use and care of the micropipettor.
Micropipettors are used
to accurately dispense small quantities of fluids. The micropipettors you will
be using are 10 microliter variable volume pipettors.
They can accurately dispense up to 10 microliters in increments of .1 microliters.
Pipettors should always be used with a pipet tip. Pipettors should never be laid down on the table top with a filled tip. Pipettors should never be held in such a way as to allow liquid in the tip to run into the shaft.
To use the pipettor, first place a plastic, disposable pipet tip on the instrument. Check the volume to be dispensed. For our experiment, the volume should be set to 10.0. The yellow operating pushbutton has two stops. The first stop is for the volume to be dispensed; the second stop, fully depressed is used to 'blow' out the sample. Depress the yellow operating pushbutton to the first stop. Insert the tip into the sample and gently release the operating pushbutton. Ten microliters of fluid will be sucked up into the pipet tip. A quick release of the button can introduce air bubbles into the sample and alter the volume in the tip. To dispense the liquid, position the tip above a well in your gel and gently depress the operating pushbutton completely. Remove the tip using the tip ejector (ejector pushbutton) or by firmly grabbing the tip and pulling to remove it from the shaft. Discard the tip; use a new pipet tip for each sample.
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The volume to be dispensed by the pipettor is indicated on the side of the pipettor. This pipettor is set to 10 microliters. The number in red indicates tenths (.1) of a microliter. The volume can be changed by rolling the calibration knobs. |
Gel electrophoresis is a technique that separates biological macromolecules based on size and electrical charge by placing these molecules in an electric field and forcing them to move through a gel matrix.
The apparatus is shown below.
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This is a picture of the apparatus you will be using. The cover is not shown. The electrodes fit into the cover/lid. The red electrode is the positive electrode or cathode. The black electrode or anode is the negative electrode. The alluminum baffles are used ONLY when pouring the gel. They block off the ends of the gel bed. The gel bed is a removable piece of plexiglass used to hold the gel while it hardens. The edges of the gel bed are notched in the center and on one end. The notches hold the gel comb. The gel comb is a plastic insert used when pouring the gel to form the sample wells. The buffer chambers hold the buffer. Buffer is essential to allow the electrical current to move through the gel. |
How to assemble the apparatus to pour the gel.
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Place gel bed on the platform in the apparatus. Notches in the sidewalls of the gel bed should be on the sides of the apparatus. The gel bed should be oriented so that the end-notched side of the bed is towards the negative (black) electrode. Place baffles at the ends of the gel bed. They should slide into formed slots. Fit the comb into notches at the end of the gel bed. Remove the agarose gel from the waterbath and pour it into the gel bed. Allow the gel to solidfy for 5 minutes. Wiggle the comb back and forth to loosen it. Then remove the comb from the gel. Remove the baffles, add buffer to the chambers and you are ready to load your samples. |
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Agarose - agarose is a highly purified form of agar. The long molecules crosslink to form a mesh network through which DNA fragments travel.
Buffer - the buffer is a solution that is the correct pH and ion concentration to allow the transmission of electrical current
Practice using the micropipettor before using your sample.
Keep the agarose in the hot water bath until you are ready to use it. It hardens very quickly.
Do not move your apparatus around once the sample has been loaded.
Do not pour a thick gel.
The gel can be brittle. Handle the poured gel carefully.
Make sure you remove the baffles.
Add sufficient buffer to fill the buffer chambers and cover the gel.