biology lab
lulsab
lab_2-_molec._techniques.docx
LAB 2- MOLECULAR BIOLOGY LAB TECHNIQUES
INTRODUCTION
This week’s lab will introduce you to three molecular biology techniques that you will use in future labs. During the course of this activity, you will be learning and practicing micropipetting, polymerase chain reaction (PCR), and DNA gel electrophoresis. Each topic below provides, or refers you to, background information on the technique prior to the hands-on activity where you will learn the technique.
Learning Objectives:
1. Be able to properly select and utilize micropipettes for the manipulation of small volumes of liquid.
2. Be able to explain how PCR amplifies DNA and be able to perform a PCR protocol.
3.
Understand how gel electrophoresis is able to separate DNA fragments, be able to pour an agarose gel, load samples, and interpret results.
Lab notebooks:
Look over the notebook guidelines posted in the general Lab Materials content folder. Begin this lab by writing a summary of the lab’s objectives.
I. Micropipettes
Pre-lab Introduction:
A micropipette is a kind of fancy eyedropper – one that comes in many different models and volume ranges. But while an eyedropper dispenses drops, micropipettes transfer microliters of fluid. Recall that ‘micro-’ is a prefix in the metric system which means “one-millionth” of the base unit (in this case, a liter, “L”). It may be easier for you to picture one milliliter (mL or ml) of water. If you mentally subdivide that milliliter of water into 1000 tiny equal-sized volumes, each volume is one microliter (abbreviated μL or μl). Watch the 2 pipetting videos posted in the lab 2 content folder ( https://www.youtube.com/watch?v=p-OPOYbeZP0 & https://www.youtube.com/watch?v=NgosWmRjjAo ) , then continue from here.
Micropipette Anatomy:
1. Examine the figures to the right to familiarize yourself with the anatomy of a micropipette.
2. Micropipette plungers have 3 positions:
a. Rest position- no pressure on plunger
b. First stop- position that will draw desired volume into tip
c. Second stop- position that will fully expel a sample from the tip
3. Pipette tips are pressed onto the “nose cone” by pressing the pipette into the tip…not pushing it on with your hand.
4. Tips are ejected after use by pressing the tip ejector button near the top of the pipette. (Note: not all pipettes have an ejector button, in which case the tip is manually removed)
5. The volume drawn by a pipette is set using the adjustment dial. This will physically move the plunger up and down as you turn the dial.
6. The volume readout will display what volume the pipette is set to draw. See the examples of volume readouts for pipettes with different volumes (our pipette colors and ranges may differ from these).
CAUTIONS When Using Micropipettes:
· Set pipette volume only within the range specified for that micropipette (e.g. 2-20 μL). Do not attempt to set a volume beyond the pipette’s min or max values, and always choose the smallest pipette that can accomodate your desired volume.
· When using a micropipette, first apply a tip. Forgetting to do this would cause liquid leakage into the nose cone. Since a micropipette works by air displacement, its internal mechanism must remain dry.
· Always keep a micropipette in a vertical position when there is fluid in the tip. Do not allow liquid to accidentally run back into the nose cone.
· Use your thumb to control the speed at which the plunger rises after taking up or ejecting fluid. Releasing the plunger too abruptly will cause liquid to pop up into the nose cone.
How to use a micropipette:
1. Check that you have the right micropipette. During this lab there may be multiple sizes of pipettes in the lab – a “P-2” for (0.2-2 μL), a “P-10” for (0.5-10 μL), a “P-20” (for 2 to 20 μL), a “P-100” (for 20-100 μL), and a “P-200” (for 20-200 μL). Check the disk on top of the plunger to see which micropipette you have.
2. Dial a desired volume. Do you understand how to read the scale? If not – ASK!
3. Push the end of the pipette into the tip, contained in a box. Press down and twist slightly.
4. Open the container from which you plan to remove liquid.
5. Hold the pipette in a vertical position. Depress the plunger to the first stop. Air equal to the volume of the setting (e.g. 10 μL) is displaced.
6. While maintaining a vertical position, immerse the tip into the liquid slightly. Do NOT put the tip too far into the liquid, this will decrease the accuracy of the measurement (see figure on bottom right).
7. Slowly release the plunger back to the rest position. Wait a second for liquid to be sucked up into the tip. The volume of liquid in the tip will equal the volume of the setting of the micropipette.
8. Have a helper open the container to which you plan to add the liquid (or use your other hand).
9. Place the tip at a slight angle (10° to 45°) against the wall of the vessel receiving the liquid, for example a well of a microwell plate. This creates a tiny adhesion effect which helps coax fluid out of the tip.
10. Depress the plunger to the first stop, wait one second, press the plunger to the second stop to expel all the liquid.
11. Move the end of the tip away from the liquid. Slowly release the plunger to the rest position.
12. Always change tips for each new reagent you need to pipette. To eject a tip, depress the ejector button while the pipette tip is in or over the disposal container.
13. If you add multiple liquids to a microcentrifuge tube, individual droplets may cling to the walls of the tube. To mix these, you can close the micro-tube and gently tap it against the table or put it in the centrifuge.
In-lab Procedure:
II. Polymerase Chain Reaction
Pre-lab Introduction:
Polymerase chain reaction is a technique that is commonly used in molecular biology to amplify a sample of DNA. It involves the use of very small volumes of water, target DNA, and 5 reactants: Buffer, forward primers, reverse primers, dNTPs, and taq polymerase. Before continuing, go online and work through the virtual PCR lab at this website: http://learn.genetics.utah.edu/content/labs/pcr/
In-lab Procedure:
Each person will prepare one PCR tube of reactants. Be sure to label your tube! Each tube will receive the recipe for the PCR reaction listed below. Record your procedure in your lab notebook, making sure to note any mistakes or problems!
Obtain a one tube of each reactant for your entire group. Keep the reactants on ice while preparing the PCR mixture. Add the following reagents to your PCR tube using the proper pipette & tips. Always change pipette tips between reagents!! BE SURE TO CHECK THAT THE PROPER VOLUME IS SET ON THE PIPETTES!
10x PCR buffer 10 l
10 M forward primer 1 l
10 M reverse primer 1 l
10 mM (2.5 mM each) dNTPs 10 l
Taq polymerase 0.5 l
Template DNA variable, ~10 ng (2 l volume today)
Water (to 100 l total) 75.5 l
Total volume 100 l
Place your tube into the PCR thermocycler. Once your group is all ready, practice turning on the pre-programmed PCR cycle, outlined below. When you are finished, you can dispose of your PCR tube in the normal trash, but please return your reagent tubes to the proper containers for other groups to use.
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PCR Program for amplification of intact bacteria |
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Temperature (oC) |
Time |
Repetitions |
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94 |
5 min |
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94 |
30 sec |
25x |
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50-55 |
30 sec |
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72 |
5 min |
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72 |
10 min |
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4 |
|
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III. Gel Electrophoresis
Pre-lab Introduction:
Gel electrophoresis is a method of separating DNA, RNA, or protein samples based upon size and electrical charge. Samples (mixed with a high-density loading dye) are loaded into “wells” in an agarose gel and electrophoresed by an electric field applied across the gel. This causes the DNA (or RNA, or protein) fragments in the samples to move from their origins (sample wells) through the gel matrix toward the positive electrode. Also separated on the gel is a DNA ladder, a sample of DNA containing a range of fragments of known length (image on right). Smaller DNA fragments migrate faster than larger ones, therefore creating a smear, or distinct bands, of DNA based upon the respective fragment sizes. You will practice gel electrophoresis this week in preparation for the real procedure next week. Before continuing, watch the online lecture about electrophoresis posted in the lab 2 content folder ( https://youtu.be/XlOglcmXNrw?t=8m16s ). This will provide you with the basic information needed to understand these instructions.
In-lab Procedure:
You will work with your group to make one agarose gel. Each person will practice loading the gel, then you will run the gel together. Record your procedure in the your lab notebook, especially any details such as whose sample is in which lane, amount of time the gel runs, etc.
CAST A GEL
1. Carefully seal the ends of a gel casting tray with tape and insert a well-forming comb. Place gel casting tray out of the way on lab bench, so that agarose poured in next step can set undisturbed.
2. Immediately prior to obtaining the agarose solution, use the designated ethidium bromide 2-20 μL pipette to add 5 μL of ethidium bromide to the center of the casting tray. This chemical is in a micro-tube wrapped in foil to keep it dark. It is also a mutagen and must be handled VERY CAREFULLY. Wear gloves whenever handling the chemical, a container that holds it, any gel that contains it, or any solution (i.e. buffer solution) that was in contact with a gel that contained ethidium bromide. Wash your hands after any possible exposure!
3. Have one group member retain the pipette tip used for the ethidium bromide for step 6.
4. Obtain a bottle of 1% agarose (50 ml) from the water bath (60oC). Microwave briefly if it is “chunky”, but be careful of hot glassware! Swirl to mix and immediately carefully pour the solution directly onto the ethidium bromide in the center of your casting tray. Pour enough agarose solution into casting tray to fill to a depth of about 5 mm. Gel should cover only about 1/3 to 1/2 the height of the comb teeth.
5. Replace the agarose bottle in the water bath while a group member continues with the following step.
6. Using the ethidium bromide pipette tip from Step 2, carefully mix the agarose solution for approximately 20 to 30 seconds to distribute the ethidium bromide stain. (you can remove the comb briefly while you mix the ethidium bromide) Also use the tip to move large bubbles or solid debris to the sides or end of the tray while gel is still liquid. Once finished, dispose of the pipette tip in the appropriately labeled waste container.
7. The gel will become cloudy as it solidifies (about 10-15 min). Do not move the casting tray while agarose is solidifying.
8. When agarose has solidified it will look dry when viewed at an angle. When this happens, unseal ends of casting tray. Dispose of tape into the proper ethidium bromide waste container.
9. Place tray in gel box so that the comb is at the negative (black) end. (DNA runs toward the red) Be careful! The gel will slip out of the end of the tray quite easily at this point.
10. If the gel box does not already have buffer solution in it from a previous group, fill it with 1x tris-acetate-EDTA (TAE) buffer solution, to a level that just covers the entire surface of the gel.
11. Gently remove the comb, being careful not to rip the wells (pull straight up).
12. Make certain that sample wells left by comb are completely submerged and filled with buffer. If “dimples” are noticed around wells, slowly add buffer until they disappear.
13. Remove any bubbles by using a pipette tip. Do not stick the end of the pipette into the wells – you might puncture the gel.
14. The gel is now ready to load with DNA.
LOAD THE GEL
Your practice gel will be loaded with a sample of loading dye mixed with water, instead of DNA. This is because DNA ladder is very expensive and unnecessary for today’s procedure. When loading a gel, make sure you draw a map of your gel in your lab notebook, to keep track of whose sample is in which lane!
1. Use a 2-20 μL micropipette to transfer 6 µL of loading dye into a micro-centrifuge tube.
2. Add 2 µL of the imitation “DNA Ladder” (water) and pipette the mixture up and down (to the first “soft” stop) several times to mix. Then change the pipette volume to 8 µL, withdraw the sample and load it into one well in the gel by doing the following:
a. Steady pipette over well using two hands and ensure there is no air at the tip of the pipette (causes improper loading). Viewing the gel from the side or end can help with proper positioning.
b. Be sure you are not pressing the plunger down, then dip the pipette tip through surface of the buffer, position it over the well. It can help to gently wiggle the pipette tip back and forth as you approach the top of the gel, so you will see and feel when you are in the well since the tip will bump into the walls of the well.
c. Slowly expel the mixture to the first (soft) stop. Carefully remove the pipette while keeping the plunger pressed down. The high density loading dye weighs down the sample, causing it to sink to the bottom of the well. Be careful not to punch tip of pipet through bottom of gel.
d. Additional group members can practice loading the gel the other wells
ELECTROPHORESIS
1. Close the top of the electrophoresis chamber and connect electrical leads to an approved power supply, anode to anode (red-red) and cathode to cathode (black-black). Make sure both electrodes are connected to same channel of power supply.
2. Turn power supply on and check that the voltage is set to ~100 V. Shortly after current is applied, loading dye can be seen moving through gel toward positive pole of the electrophoresis chamber.
3. Allow the DNA to electrophorese until the blue bands nears the red band across the gel (approx. 20-40 minutes). While you wait, you can practice your pipetting more!
4. Turn off power supply, disconnect leads from the inputs, and remove top of electrophoresis chamber.
5. Carefully remove the casting tray while wearing gloves and slide gel into a plastic ZipLock bag. Do this over a large weighing tray to prevent ethidium bromide from spreading. Leave the TAE solution in the electrophoresis chamber.
6. Take your gel to the the UV lightbox, and place it on top, and turn the light on. Be sure to wear goggles so the UV light doesn’t damage your eyes! You will not see bands, since there was no DNA in the gel, but the ethidium bromide should have an orang-ish glow. See the example image to the right: the DNA ladder is in the bottom row, with other DNA samples in the top 4 rows. You can still see the loading dye, further to the right, and wells to the left.
7. Draw the bands from your lane onto the gel map below. Also take a picture of your gel to be sure you have a record of the location of the bands.
8. Dispose of your gel, bag, and gloves in the proper ethidium bromide waste container.
IV. Conclusion
In your lab notebook, write a brief (1 paragraph) summary of what you did in the lab and reflect on which procedures were the most challenging, or which skills you may need to practice further.
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