DNA Electrophoresis (Lisa Hellinger, Shayleen Scantlin)
Authors:
Lisa Hellinger (AP Biology Teacher, Immaculate Heart High School)
Shayleen Scantlin (Biology and AP Biology Teacher at Panorama High School)
Principles:
Gel electrophoresis equipment uses an electrical charge to separate negatively charged DNA fragments by size.
Standards:
NGSS Science & Engineering Standards
SEP 3 - Planning and carrying out investigations
Planning and carrying out investigations in 9-12 builds on K–8 experiences and progresses to include investigations that provide evidence for and test conceptual, mathematical, physical, and empirical models
NGSS Cross-cutting Concept Standards
HS-LS4
Different patterns may be observed at each of the scales at which a system is studied and can provide evidence for causality in explanations of phenomena. (HS-LS4-1), (HS-LS4-3)
NGSS Disciplinary Core Idea Standards
LS1.A: Structure and Function
All cells contain genetic information in the form of DNA molecules. Genes are regions in the DNA that contain the instructions that code for the formation of proteins, which carry out most of the work of cells. (HS-LS1-1) (HS-LS3-1)
LS4.A: Evidence of Common Ancestry and Diversity
Genetic information provides evidence of evolution. DNA sequences vary among species, but there are many overlaps; in fact, the ongoing branching that produces multiple lines of descent can be inferred by comparing the DNA sequences of different organisms. Such information is also derivable from the similarities and differences in amino acid sequences and from anatomical and embryological evidence. (HS-LS4-1)
LS3.A: Inheritance of Traits
Each chromosome consists of a single very long DNA molecule, and each gene on the chromosome is a particular segment of that DNA. The instructions for forming species’ characteristics are carried in DNA. All cells in an organism have the same genetic content, but the genes used (expressed) by the cell may be regulated in different ways. Not all DNA codes for a protein; some segments of DNA are involved in regulatory or structural functions, and some have no as-yet known function. (HS-LS3-1)
Pipetting and Loading Practice Procedure:
Materials Needed:
5 - microtubes with blue, red, yellow, green, clear solution
1 - .5 - 10 ul pipette
1 - 5 - 50 ul pipette
3 - empty microtubes for each student
1 - tray of practice pipette tips
1 - microtube of loading dye
1 - practice gel submerged in tray of water
Pipetting Practice:
Send one team member to collect materials
Using the small pipette, set it to the required setting and add the following: CAUTION! Change pipette tips between different solutions to avoid contamination. Place used tips, to be washed and saved, in the plastic cup. Do not place solutions on bottom of the microtube or to near the lid. “Hang” the droplets on the side of the wall without letting them touch.
4 ul of yellow solution to all microtubes. (This simulates the DNA)
5 ul of red solution to all microtubes. (This simulates the buffer)
1 ul of clear solution to only one microtube. (This simulates an enzyme)
1 ul of green solution to only one microtube. (This simulates an enzyme)
1 ul of blue solution to only one microtube. (This simulates an enzyme)
Centrifuge your microtubes in order to mix the contents. CAUTION! Make sure the centrifuge is balanced or it will break! When you perform the lab the reaction between the DNA and the restriction enzymes will not begin until you centrifuge and place them in the waterbath.
Add 2 ul of loading dye and centrifuge again to mix in the dye.
How many total ul were added to your microtube? ________
Set your large pipette to 10 ul.
Withdraw 10 ul from your tube. Place the pipette tip at the bottom of the tube and very slowly withdraw the sample. The trick to this is to not let the tip of the pipette tip come off the bottom of the the tube even after you have released your thumb. The final drop will be pulled into the tip at the last second. If you pull the tip away too early you will get air. There should be no air bubble at the end of your tip.
Loading Practice:
When you have mastered step seven practice loading your sample into the gel.
NO NO YES!
Using two hands, steady the pipette over the well you are going to load. Expel any air in the end of the pipette before loading the DNA sample.
Dip the pipette tip through the surface of the buffer, position it just inside the well, and slowly expel the mixture. Sucrose in the loading dye weighs down the sample, causing it to sink to the bottom of the well. Be careful not to puncture the bottom of the well with the pipette tip or re-aspirate your sample up into the pipette by letting your thumb release the plunger.
When you are finished rinse out your microtubes. Leave the lids open, and place in the container designated for used microtubes and place practice gel on cart to dry.
Investigation Procedure:
Materials Needed:
250 mL of TBE Buffer
2.85 L of Distilled water
1% agarose solution
Water bath or microwave
Flask
CarolinaBLU stain
comb
gel box
DNA samples
masking tape
gel electrophoresis chamber
power supply
needle point pipets
light box
Preparing the Agarose Gel
• Measure 1.25 g Agarose powder and add it to a 500 ml flask
• Add 125 ml TAE Buffer to the flask. (the total gel volume well vary depending on the size of the casting tray)
• Melt the agarose in a microwave or hot water bath until the solution becomes clear. (if using a microwave, heat the solution for several short intervals - do not let the solution boil for long periods as it may boil out of the flask).
• Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly.
• Seal the ends of the casting tray with two layers of tape.
• Place the combs in the gel casting tray.
• Pour the melted agarose solution into the casting tray and let cool until it is solid (it should appear milky white).
• Carefully pull out the combs and remove the tape.
• Place the gel in the electrophoresis chamber.
• Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel.
Loading the Gel
• Add 6 ml of 6X Sample Loading Buffer to each 25 ml PCR reaction
• Record the order each sample will be loaded into the gel, including who prepared the sample, the DNA template - what organism the DNA came from, controls and ladder.
• Carefully pipette 20 ml of each DNA sample into separate wells in the gel.
• Pipette 10 ml of the DNA ladder standard into at least one well of each gel.
Running the Gel
• Place the lid on the gel box, connecting the electrodes.
• Connect the electrode wires to the power supply, making sure the positive (red) and negative (black) are correctly connected. (Remember – “Run to Red”)
• Turn on the power supply to about 100 volts. Maximum allowed voltage will vary depending on the size of the electrophoresis chamber – it should not exceed 5 volts/ cm between electrodes!
• Let the power run until the blue dye approaches the end of the gel.
• Turn off the power.
• Disconnect the wires from the power supply.
• Remove the lid of the electrophoresis chamber.
• Using gloves, carefully remove the tray and gel.
Gel Staining
• Using latex or nitrile gloves, remove the gel from the casting tray and place into the staining dish.
• Add warmed (50-55°) staining mix.
• Allow gel to stain for at least 25-30 minutes (the entire gel will become dark blue).
• Pour off the stain (the stain can be saved for future use).
• Rinse the gel and staining tray with water to remove residual stain.
• Fill the tray with warm tap water (50-55°). Change the water several times as it turns blue. Gradually the gel will become lighter, leaving only dark blue DNA bands.
Destain completely overnight for best results.
Gel Viewing
• View the gel against a white light box or bright surface.
• Record the data while the gel is fresh, very light bands may be difficult to see with time.
Explanation:
During electrophoresis, the gel is submerged in a chamber containing a buffer solution and a positive and negative electrode. Under an electrical field, DNA is negative and will move to the positive electrode (red) and away from the negative electrode (black). The DNA can be visualized by the use of a dye that binds to the DNA fragments. The smaller pieces will travel farther and the longer pieces will be closer to the well.
Organisms have different lengths of DNA fragments because each individual has a different sequence of nucleotides. Differences in DNA sequences will change where the restriction enzyme cuts; therefore; creating different sized fragments of DNA. The different sizes will sort themselves according to their lengths and create the different patterns like the one found in the gel to the left.
In this example it seems the victim's blood matches the blood found on the suspects shirt.
Questions:
1. If an increased concentration of agarose gel were used, how would it affect the separation of the bands?
The tighter gel matrix reduces effective separation of larger fragments but will separate the smaller DNA fragments more effectively.
2. If the two suspects had been identical twins, how would it have influenced the results?
The two suspects would be so alike that it might not be possible to determine which one committed the crime.
3. If more than two lanes of bands look alike, what may have happened during the gel-loading?
Someone may have forgotten to change pipet tips between samples which would contaminate the DNA in one well.
4. Why do some DNA fragments move farther than others?
The shorter fragments will move farther because they are smaller and pass through the gel easier. The gel acts like a sieve.
5. Why do people have differences in their band pattern?
Differences at where the restriction enzymes cut.
Uses of DNA Electrophoresis:
Forensics - compare a suspects DNA to DNA found at a crime scene or on a victim
Genetics - help determine paternity, identify remains from an accident
Conservation Biology - help determine the illegal trade of endangered species
Microbiology - identify pathogens
Medicine - to determine if a patient carries a gene for a particular disease
Archaeology - to learn about past human activity
Evolution - to determine evolutionary relationships between species
