Name: dna isolation and restriction enzyme analysis
Uploaded: 2019-01-31T09:26:45.000Z
Duration: 4 min 46 s
Channel: JoVE

K12 Standards

north-carolina-k-12-science-standard-course-of-study

North Carolina K-12 Science Standard Course of Study

9-12

Biology

heredity-inheritance-and-variation-of-traits

Heredity- Inheritance and Variation of Traits

LS.Bio.8.1

dna-isolation-and-restriction-enzyme-analysis

DNA Isolation and Restriction Enzyme Analysis

lab manual sub articles

Lab Manual Sub Articles

<p>DNA extraction is the removal and purification of DNA from cells. First, cells are lysed - broken open - usually though a combination of physical disruption and treatment with chemicals, such a detergent which dissolves the cell and nuclear membranes. SDS or sodium dodecyl sulfate is a commonly use detergent that works by solubilizing the proteins and lipids that make up these membranes. The contents can then float freely into the surrounding solution. </p>

<p>Next, DNA must be separated from the other molecules present. Proteinase K added to the reaction will break down peptide bonds and digest contaminating proteins. Salt is then added to the sample, which stabilizes the negatively charged phosphate groups in the backbone of the DNA and, after the addition of ice cold alcohol, precipitates the DNA out of solution. The white precipitate is collected by spinning it in a centrifuge where it settles to the bottom of the tube. After washing and resuspending it in a buffer solution, the extracted DNA can finally be used in research or biotechnology applications. </p>

<p>Some biotechnology applications, like DNA fingerprinting, which can identify novel patterns in DNA specific to an individual or individuals, involve the use of restriction enzymes. Restriction enzymes are molecules which interact with DNA and recognize specific sequences. Once their specific site is identified, they cut the DNA. This will result in the strand being cut into one or more linear pieces. If the DNA extracted was a plasmid, a circular piece of DNA most often found in bacteria, any cuts will result in the DNA forming a linear fragment or fragments. Different restriction enzymes recognize different DNA sequences, so using a combination of these can result in distinct fragments being produced. </p>

<p>In DNA fingerprinting, we can then examine these fragments using a technique called DNA or gel electrophoresis. To prepare gels, powdered agarose is mixed with a buffer and heated until dissolved. A nucleotide stain is then added to the warm mixture and then this solution is poured into the casting mold. A comb is inserted to form the wells. When solidified, the gel is transferred to a gel box filled with buffer and the comb is removed. A reference mixture of dyed DNA fragments of known lengths, the DNA ladder, is added to one well and the dyed DNA samples of interest are loaded into the remaining wells. The box is connected to a power source and switching the power on induces the migration of the negatively charged phosphate groups in DNA nucleotides through the gel towards the anode, the positive end. Smaller pieces move more quickly than the larger fragments, which migrate with difficulty. </p>

<p>When the run is complete the gel is exposed to ultra violet light to visualize the nucleotide stain in the DNA samples. Their presence can be confirmed based on their relative location to the ladder bands. Because DNA with different sequences will have cut sites at different locations, this can produce novel band patterns, or fingerprints, which can be used to distinguish individuals or variant DNA profiles. </p>

<p>In this lab you will perform DNA extractions using a buffer with and without SDS to assess the importance of detergents in DNA isolation and then digest plasmid DNA with different restriction enzymes to examine the resulting DNA profiles.</p>


dna isolation and restriction enzyme analysis

<p>The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our understanding of ourselves and other organisms. DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and forensics.</p>

<h4>Isolating DNA from Cells</h4>

<p>DNA isolation is a fairly simple procedure that requires few but essential steps: cell harvesting, lysis, protein degradation, and finally DNA precipitation. Generally, DNA can be harvested from small amounts of tissue, such as a single leaf of a plant, hair follicles of animals, or epithelial cells that can be easily sloughed off within the mouth with a simple saline solution. Since DNA is compartmentalized inside the nucleus or mitochondria within eukaryotic cells, scientists first break apart the cell and nuclear membrane to expose the DNA, which is done with the lysis step. However, once DNA is exposed, it can be degraded by heavy metal ions and extremes of pH. Therefore, a detergent-containing buffer solution is used to not only dissolve the cell membrane, but also to protect DNA from degrading elements in the solution. Moreover, DNA is an unusually long molecule that is condensed inside the nucleus by packing tightly around special proteins called histones. Therefore, an enzyme called Proteinase K, which degrades peptide bonds of proteins, is added to separate the DNA from histones and other proteins. Next, scientists separate DNA from the rest of the cellular extract by using its chemical properties. Due to the negatively charged structure of nucleic acids, Na<sup>+</sup> ions in a sodium chloride solution bind to and integrate into entangled DNA strands. Once the DNA is clumped, it can be observed by the naked eye. DNA is insoluble in alcohol and readily precipitates at low temperatures, thus scientists add cold alcohol to precipitate DNA. Finally, the precipitated DNA can be dissolved and stored in deionized water or a mild buffer, free of any enzymes that can degrade nucleic acids, until it is used.</p>

<h4>Quantifying DNA</h4>

<p>Reactions that use DNA often require high purity and precise amounts of DNA. Therefore, following DNA isolation, scientists use a spectrophotometer to quantify the amount of DNA in their sample, as well as the purity of the DNA in the sample. A spectrophotometer measures the intensity of a light beam passed through a sample as a function of its wavelength. Nucleic acids absorb light at 260 nm, whereas proteins absorb light at 280 nm. By measuring the intensity of a light beam at 260 nm, scientists can determine the DNA concentration. Furthermore, by assessing the light intensity at both 260 nm and 280 nm, the purity of the DNA content can be determined. In a sample of pure DNA, the 260/280 nm ratio should approach the value of 2.</p>

<h4>Restriction Enzymes</h4>

<p>For a number of analyses, such as cloning, sequencing, and mapping DNA fragments and genomes, the isolated DNA needs to be cut at specific sequences using restriction enzymes. These enzymes are produced by bacteria as mechanisms of innate immunity against intruding viral DNA and function as molecular scissors. Hence, after restriction enzymes identify a viral DNA, they start to break it apart by cutting it into segments at certain nucleotide sequences they recognize. These nucleotide sequences are often six to twelve nucleotides long and are generally palindromic, which means that they have the same nucleotide sequence in the 3&#8217;-5&#8217; and 5&#8217;-3&#8217; directions. For instance, the restriction enzyme EcoRI cuts DNA at the sequence 3&#8217;...GAATTC&#8230;-5&#8217;, which reads the same in the 5&#8217;-3&#8217; direction. Like EcoRI, there are hundreds of enzymes that correspond to many distinct palindromic sequences. Once DNA is cut by these enzymes, scientists can load the DNA onto a gel in an electrophoresis chamber and pass electrical current through the gel. DNA carries negative charges, which causes DNA fragments to migrate towards the anode. However, the pores of the gel slow down larger DNA fragments relative to smaller DNA sequences, thus the pieces separate according to their size. After the completion of the electrophoresis, DNA fragments can be visualized with the aid of dyes that specifically bind to DNA molecules.</p>

<p>Cutting DNA with restriction enzymes and separating DNA fragments with electrophoresis allows researchers to identify the sizes of unknown fragments relative to a standard ladder. These fragments of interest can then be further isolated and ligated into a plasmid vector for genetic cloning. Genetic engineering and laboratory analyses have been improved immensely with these methods. With DNA profiling, scientists are able to sequence genomes to further understand the workings of organisms and are also able to identify and characterize certain genes<sup>1</sup>.</p>

<p>DNA isolation and profiling are also practical for events directly impacting everyday life. For example, forensics scientists routinely analyze DNA samples to provide evidence for criminal and civil cases<sup>2</sup>. Similarly, healthcare workers extract and analyze DNA to check for paternity and genetic diseases. Recently, DNA ancestry analysis kits became popular by allowing users determine their genetic lineage, find relatives, and even discover their genetic disposition to various health conditions<sup>3</sup>. Finally, with advancements in biotechnology and genetic engineering, personalized medicine is gaining traction among clinical researchers to develop treatments by using an individual&#8217;s genetic data, paving the way to effectively treat patients without detrimental side effects<sup>4</sup>.</p>

<h4>References</h4>

<ol class="references">
	<li><strong>Chambers, GK, et al.</strong> DNA fingerprinting in zoology: past, present, future. <em>Investig Genet. </em>2014, Vol. 5, 3 doi: 10.1186/2041-2223-5-3.</li>
	<li><strong>Roewer, L.</strong> DNA fingerprinting in forensics: past, present, future. <em>Investig Genet. . </em>2013, Vol. 4, 22 doi: 10.1186/2041-2223-4-22.</li>
	<li><strong>Kirkpatrick, BE and Rashkin, MD.</strong> Ancestry Testing and the Practice of Genetic Counseling. <em>J Gen Counseling. </em>2016, Vol. 26, 1 (6-20).</li>
	<li><strong>Hamburg, MA and Collins, FS.</strong> The Path to Personalized Medicine. <em>N Engl J Med . </em>2010; , Vol. 363, 301-304.</li>
</ol>


The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our ...

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Learn how DNA is isolated, measured with a spectrophotometer, cut with restriction enzymes, and separated by gel electrophoresis for analysis.

DNA Extraction, Quantification, and Cutting | JoVE K12

Prepare the materials, buffers, saline, and ice-cold ethanol needed for a high school DNA extraction and restriction enzyme lab.

Preparing Reagents for DNA Extraction Lab | JoVE K12

Compare cheek cell DNA isolation with and without SDS, then use virtual and lab restriction digests of lambda DNA to study gel band patterns.

Cheek Cell DNA and Gel Band Patterns | JoVE K12

<ul><li><a href="#" data-time="0.0">Preparation of Solutions and Materials
</a></li></ul>

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Preparation of Solutions and Materials
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: For this lab to run smoothly, it is recommended to not exceed 12 samples per laboratory class.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">First gather the necessary materials, including popsicle sticks, funnels, 15 mL conical tubes, micropipettes, ice buckets with ice, small cups and microcentrifuge tubes.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next set the water bath used in part one of the laboratory exercise, to 56 &#176;C.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Set a second water bath to be used in part two of the laboratory exercise to 37 &#176;C.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To make TE buffer, first measure out 400 mL of RO water.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Then add five mL of 1M Tris-HCL pH 8, and then 1 mL of 0.5 M EDTA solution.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Next, add sufficient RO water to bring the solution up to 500 mL.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">To make TE buffer with SDS or TES buffer, again measure out 400 mL of RO water.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Add five milliliters of 1M Tris-HCL pH 8, and then add 1 mL of 0.5 molar EDTA solution.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Now add 5 mL of 10% SDS solution.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Bring the volume up to 500 mL, using RO water.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Next, prepare the 1% saline solution that the students will use to collect cheek cells. In an empty, clean soda bottle, dissolve 5 g of NaCl in 500 mL of RO water.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Pour 10 mL aliquots into small drinking cups for the students to use, one per student group.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">After this, prepare a sufficient quantity of 10 mL aliquots of 100% ethanol for the students to use. NOTE: Store these ethanol aliquots in the freezer or refrigerator to keep them preferably ice-cold.
</div></li></ul></li></ol>

<div class="section-question"><h4 class="question-sect">What are the essential steps of DNA isolation?</h4><p>The essential steps of DNA isolation are cell harvesting, lysis, protein degradation, and DNA precipitation.
</p></div><div class="section-question"><h4 class="question-sect">How is the quantity and purity of DNA determined?</h4><p>Scientists use a spectrophotometer, which measures the intensity of a light beam passed through a sample as a function of its wavelength. By quantifying the output of wavelengths that are specific to nucleic acids or impurities, one can not only quantify the amount of DNA but also estimate the purity of the DNA sample.
</p></div><div class="section-question"><h4 class="question-sect">What is a restriction enzyme?</h4><p>It is an enzyme of bacterial origin that can recognize specific DNA sequences and cut it into segments at those locations. 
</p></div><div class="section-question"><h4 class="question-sect">How is gel electrophoresis used for DNA analysis?</h4><p>Scientists can load DNA onto a gel in an electrophoresis chamber and pass electrical current through the gel. DNA carries negative charges, which causes DNA fragments to migrate towards the anode. However, the pores of the gel slow down larger DNA fragments relative to smaller DNA sequences, thus the pieces separate according to their size.
</p></div><div class="section-question"><h4 class="question-sect">Why is genetic profiling important?</h4><p>DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and forensics. In addition, these advancements recently paved the way for personalized medicine with improved treatment outcomes.
</p></div>

Preparation of Solutions and Materials
ExpandNOTE: For this lab to run smoothly, it is recommended to not exceed 12 samples per laboratory class.
First ...

<p>For this lab to run smoothly, it is recommended to not exceed 12 samples per laboratory class. First gather the necessary materials, including popsicle sticks, funnels, 15 milliliter conical tubes, micropipettes, ice buckets with ice, small cups and microcentrifuge tubes. Next set the water bath used in part one of the laboratory exercise, to 56 degrees Celsius.</p><p>Set a second water bath to be used in part two of the laboratory exercise to 37 degrees Celsius. To make TE buffer, first measure out 400 milliliters of RO water. Then add five milliliters of one molar Tris-HCL with a PH of eight, and then one milliliter of 0.5 molar EDTA solution, then add sufficient RO water to bring the solution up to 500 milliliters.</p><p>To make TE buffer with SDS or TES buffer, again, measure out 400 milliliters of RO water. Then add five milliliters of one molar Tris-HCL with a PH of eight, one milliliter of 0.5 molar EDTA solution, and five milliliters of 10%SDS solution. Bring the volume up to 500 milliliters, using RO water.</p><p>Prepare the 1%saline solution that the students will use to collect cheek cells. In an empty, clean soda bottle, dissolve five grams of sodium chloride in 500 milliliters of RO water. Pour 10 milliliter aliquots into small drinking cups for the students to use, one per student.</p><p>After this prepare a sufficient quantity of 10 milliliter aliquots of 100%ethanol for the students to use. Please take time to review the material list and the Student Protocol video, to be fully prepped for the lab.</p>

<ul><li><a href="#" data-time="0.0">DNA Isolation
</a></li><li><a href="#" data-time="323">Restriction Enzyme Virtual Digest
</a></li><li><a href="#" data-time="470">DNA Profiling
</a></li><li><a href="#" data-time="745">Results
</a></li></ul>

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">DNA Isolation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this experiment you will perform DNA isolation under two experimental conditions: one using a buffer containing the detergent SDS and one without detergent. Hypotheses: The alternate hypothesis for this experiment might be that the sample prepared without SDS, a strong anionic detergent that breaks apart cell membranes, will yield less DNA than the sample prepared with SDS. The null hypothesis for this experiment might be that both samples will yield an equal amount of DNA.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To begin collect a tissue sample for DNA isolation. Here you will be using your own cheek cells. Use a Popsicle stick to scrape the insides of your cheeks for approximately 30 s. IMPORTANT: Do not scrape hard enough to cause pain or draw blood.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Position a funnel in a 15-mL conical tube. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then take 10 mL of the 1% saline solution provided to you in a labeled cup and swish it around in your mouth without swallowing. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Continue swishing for 90 s and then spit the saline solution containing your cheek cells into the funnel to collect in the conical tube.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Next, label two empty microcentrifuge tubes with your initials. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Label one of the tubes as +SDS and the other as -SDS to differentiate the two treatments. 
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Then add 1.5 to 2 mL of the saline solution containing the cheek cells to each tube.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Spin the tubes in a microcentrifuge for 30 s. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Remove the tubes from the microcentrifuge and carefully pour off the supernatant without disturbing the cells collected at the bottom of each tube. 
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Fill the tubes with more of the saline solution containing the cheek cells and repeat the centrifugation. 
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Repeat this process taking care to evenly distribute cells between the +SDS and -SDS tubes until all of the cheek cells have been collected.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, add 1 mL of lysis buffer containing SDS to the tube marked as +SDS, and then resuspend the cell pellet by pipetting up and down. 
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then add 1 mL of lysis buffer without SDS to the tube marked with -SDS and resuspend the cell pellet by pipetting up and down.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">After this add 10 &#181;L of proteinase K to each tube and then carefully place the tubes in the 56 &#186;C water bath for 10 min. 
</div></li><li data-sub-note-item="1-16"><div class="lab-step">After 10 minutes carefully remove the tubes from the water bath and add 100 &#181;L of 2.5 M NaCl to each tube. 
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Close the tubes firmly and invert several times to mix.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Next, label one 15 mL conical tube as +SDS and another with -SDS. 
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Pour the contents of each microcentrifuge tube into the corresponding 15 mL conical tube. 
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Obtain one 10 mL aliquot of 100% ethanol from the freezer. 
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Hold one of the conical tubes at an angle and slowly dispense 1 mL of 100% ethanol into the tube to form a layer on top of the liquid already in the tube.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Repeat this process for the second conical tube. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Now look at both of your tubes. There should be a stringy precipitate present at the interface of the ethanol and the aqueous layer below it. This stringy material contains the isolated DNA. Take pictures of the DNA in the tube and make clear descriptive notes and drawings of your observations in your notebook. 
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Using a glass stir rod, pick up the DNA from the +SDS tube and examine it. Repeat this for the -SDS tube, again recording your observations. Take note of whether one of the tubes appears to have more precipitate than the other, and why this might be.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Finally, discard all liquid waste into the appropriate waste container and return all tools to their proper locations in the lab.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Restriction Enzyme Virtual Digest
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To study how restriction enzymes work, first navigate to the New England Biolabs website: <a href="https://www.neb.com/" target="__blank">https://www.neb.com/</a>
</div></li><li data-sub-note-item="2-2"><div class="lab-step">The resources available at this site allow researchers to run virtual experiments before they are performed in the lab. Click on &#8220;Tools and Resources&#8221; to find the NEBcutter tool and open the program.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To the right of the search box find the list that says &#8220;Viral + phage&#8221;. From that list select Lambda. This is the name of the DNA you will be digesting. Select the Linear option where the tool says &#8220;the sequence is&#8221; and the &#8220;NEB enzymes&#8221; option where the tool says, &#8220;Enzymes to use&#8221;. Then click Submit.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">The next page will display a full restriction map of the lambda DNA. This restriction map can be used to predict what will happen if certain enzymes are used. Next, click on &#8220;Custom digest&#8221; under the &#8220;Main options&#8221; heading.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Search for the enzyme StuI and note that it works optimally in buffer 2.1 or CS buffer. Select the StuI enzyme and click digest.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">In the next window click on &#8220;View gel&#8221; under the &#8220;Main options&#8221; header. Select the option to run a virtual 1% gel using a pBR322-BstN1 digest.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">After the results appear, take a screenshot and paste it into a blank document.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">After this, navigate back to the main page of the NEBcutter tool and select &#8220;New custom digest&#8221;.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">This time search for the BsrGI enzyme and note that it performs optimally in buffer 2.1 or buffer 3.1. Select the enzyme and click digest.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Under the Main options header click on &#8220;View gel&#8221; and select the options to run a virtual 1% gel using a pBR322-BstN1 digest.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Take a screenshot of the results and save this in a text document.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Finally, repeat these steps using both StuI and BsrGI together in a single digest.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Again, take a screenshot and save the resulting gel.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">DNA Profiling
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Before starting the restriction enzyme digests first put your aliquots of lambda DNA, NEB buffer 2.1, StuI enzyme, and BsrGI enzyme into an ice bucket, and take this to your work area.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Label a tube as StuI &#8211; this will contain the StuI digest. 
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Label a tube as BsrGI to contain the BsrGI digest. 
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Then, label a tube S+B, to contain the digest with both of the enzymes.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Use the appropriate micropipetters to set up the reactions listed in the Table 1:<a href="https://www.jove.com/CDNSource/lm/labs/12/Lab_12_Table_1.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="3-6"><div class="lab-step">First, pipette the 40 &#181;L of water into each tube.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Then, using a fresh pipette tip each time, dispense 5 &#181;L of buffer 2.1 into each tube and pipette up and down to mix.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Next, add 4 &#181;L of lambda DNA to each tube, and then pipette 0.5 &#181;L of each enzyme into the appropriate tubes.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Finally, add a sufficient amount of water to each tube to bring the total volume up to 50 &#181;L.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">Next, incubate the tubes at 37 &#186;C for 15 - 60 min in a water bath, and return the buffers and enzymes to the freezer.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">To prepare an agarose gel to run the digest, in a 250 mL flask mix 1 g of agarose powder with 100 mL of TAE buffer.
</div></li><li data-sub-note-item="3-12"><div class="lab-step">Microwave the solution for approximately 90 s at full power, taking care to prevent the solution from boiling over.
</div></li><li data-sub-note-item="3-13"><div class="lab-step">Next, carefully remove the solution from the microwave using a glove or heat pad and place it to cool for about 15 min. 
</div></li><li data-sub-note-item="3-14"><div class="lab-step">While the agarose solution cools set up a casting tray. 
</div></li><li data-sub-note-item="3-15"><div class="lab-step">When the agarose solution is cool add 5 &#181;L of 10,000X SYBR Safe gel stain to the solution.
</div></li><li data-sub-note-item="3-16"><div class="lab-step">Add the comb which is used to make the wells of the gel to the casting tray of the gel electrophoresis apparatus.
</div></li><li data-sub-note-item="3-17"><div class="lab-step">Pour the agarose gel solution into the casting tray and allow it to solidify. 
</div></li><li data-sub-note-item="3-18"><div class="lab-step">Once the gel is solid, pull the comb straight up to remove it from the gel, and then remove the dams from the casting tray if appropriate.
</div></li><li data-sub-note-item="3-19"><div class="lab-step">Place the gel in the electrophoresis chamber with the wells positioned closest to the negative or black electrode and fill the chamber with 1x TAE buffer to completely cover the gel.
</div></li><li data-sub-note-item="3-20"><div class="lab-step">Remove your samples from the water bath.
</div></li><li data-sub-note-item="3-21"><div class="lab-step">Then add 8.3 &#181;L of 6X loading dye to each sample.
</div></li><li data-sub-note-item="3-22"><div class="lab-step">Dispense 10 &#181;L of the pBR322-BstN1 digest which contains DNA fragments of known size into the first well.
</div></li><li data-sub-note-item="3-23"><div class="lab-step">Then load 25 &#181;L of sample StuI into the second well.
</div></li><li data-sub-note-item="3-24"><div class="lab-step">Place 25 &#181;L of sample BsRGI into the third well.
</div></li><li data-sub-note-item="3-25"><div class="lab-step">Load 25 &#181;L of sample S+B into the fourth well.
</div></li><li data-sub-note-item="3-26"><div class="lab-step">To run the gel plug the leads into the electrophoresis chamber and then into the power supply. The red positive lead should be at the end farthest away from the wells and the black negative lead should be nearest to the wells. This is because the samples will run from negative to positive.
</div></li><li data-sub-note-item="3-27"><div class="lab-step">Turn on the power supply and set the gel box to run at 170 volts for approximately 60 minutes.
</div></li><li data-sub-note-item="3-28"><div class="lab-step">After the run is complete turn off the power to the gel box and disconnect the electrodes from the tank.
</div></li><li data-sub-note-item="3-29"><div class="lab-step">Then remove the gel from the electrophoresis chamber and observe it with a UV transilluminator.  CAUTION: Protect eyes from the UV by wearing goggles or using the provided shield.
</div></li><li data-sub-note-item="3-30"><div class="lab-step">Finally, use a cell phone camera to take a picture of the gel from directly above and parallel to the gel surface.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">Compare the gel you ran in the lab to your virtual enzymatic digest with StuI.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">The virtual digest with StuI resulted in seven bands. Count the bands in the StuI lane of your gel and note whether this matched the virtual digest or appeared to show a similar pattern. NOTE: Small bands on agarose electrophoresis gels are often invisible.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Now look at the results for your BsrGI digest. The virtual digest with BsrGI resulted in six bands. Count the bands in the BsrGI lane. Again, note whether the virtual digest and lab digest showed the same or a similar pattern.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Finally, look at the virtual digest with StuI and BsrGI. The virtual digest with both enzymes resulted in 12 bands. Count the bands in the corresponding lane of your gel, and consider whether the DNA banding pattern looks similar to the virtual digest.
</div></li></ul></li></ol>

DNA Isolation
ExpandNOTE: In this experiment you will perform DNA isolation under two experimental conditions: one using a buffer containing the detergent ...

<p>In this experiment you will perform DNA isolation under two experimental conditions:one using a buffer containing the detergent SDS and one without detergent. The alternate hypothesis for this experiment might be that the sample prepared without SDS, a strong anionic detergent that breaks apart cell membranes, will yield less DNA than the sample prepared with SDS. The null hypothesis for this experiment might be that both samples will yield an equal amount of DNA.</p><p>To begin collect a tissue sample for DNA isolation. Here you will be using your own cheek cells. Use a Popsicle stick to scrape the insides of your cheeks for approximately 30 seconds.</p><p>Position a funnel in a 15-milliliter conical tube. Then take 10 milliliters of the 1%saline solution provided to you in a labeled cup and swish it around in your mouth without swallowing. Continue swishing for 90 seconds and then spit the saline solution containing your cheek cells into the funnel to collect in the conical tube.</p><p>Next label two empty microcentrifuge tubes with your initials. Label one of the tubes as plus SDS and the other as minus SDS to differentiate the two treatments. Then add 1.5 to two milliliters of the saline solution containing the cheek cells to each tube.</p><p>Spin the tubes in a microcentrifuge for 30 seconds. Remove the tubes from the microcentrifuge and carefully pour off the supernatant without disturbing the cells collected at the bottom of each tube. Fill the tubes with more of the saline solution containing the cheek cells and repeat the centrifugation.</p><p>Repeat this process taking care to evenly distribute cells between the SDS-positive and SDS-negative tubes until all of the cheek cells have been collected. Next add one milliliter of lysis buffer containing SDS to the tube marked as plus SDS, and then resuspend the cell pellet by pipetting up and down. Then add one milliliter of lysis buffer without SDS to the tube marked with minus SDS, and resuspend the cell pellet by pipetting up and down.</p><p>After this add 10 microliters of proteinase K to each tube and then carefully place the tubes in the 56 degrees Celsius water bath for 10 minutes. After 10 minutes carefully remove the tubes from the water bath and add 100 microliters of 2.5 molar sodium chloride to each tube. Close the tubes firmly and invert several times to mix.</p><p>Next, label one 15-milliliter conical tube as plus SDS and another with minus SDS. Then pour the contents of each microcentrifuge tube into the corresponding 15-milliliter conical tube. Obtain one 10-milliliter aliquot of 100%ethanol from the freezer.</p><p>Hold one of the conical tubes at an angle and slowly dispense one milliliter of 100%ethanol into the tube to form a layer on top of the liquid already in the tube. Repeat this process for the second conical tube. Now look at both of your tubes.</p><p>There should be a stringy precipitate present at the interface of the ethanol and the aqueous layer below it. This stringy material contains the isolated DNA. Take pictures of the DNA in the tube and make clear descriptive notes and drawings of your observations in your notebook.</p><p>Then using a glass stir rod pick up the DNA from the SDS-positive tube and examine it. Repeat this for the SDS-negative tube, again recording your observations. Now without looking at the labels observe both of your samples.</p><p>Do the tubes appear to contain the same amount of precipitated DNA? Does one tube have more precipitated DNA than the other? Using what you have learned about DNA extraction and SDS, can you determine which sample was prepared with SDS and which sample was prepared without SDS?</p><p>Finally, discard all liquid waste into the appropriate waste container and return all tools to their proper locations in the lab. To study how restriction enzymes work first navigate to the website listed on the screen. The resources available at this site allow researchers to run virtual experiments before they are performed in the lab.</p><p>Click on Tools and Resources to find the NEBcutter tool. Open the program. Then to the right of the search box find the list that says Viral phage.</p><p>From that list select Lambda. This is the name of the DNA you will be digesting. Select the Linear option where the tool says, The sequence is and the NEB enzymes option where the tool says, Enzymes to use.</p><p>Then click Submit. The next page will display a full restriction map of the lambda DNA. This restriction map can be used to predict what will happen if certain enzymes are used.</p><p>Next, click on Custom digest under the Main options heading. Search for the enzyme Stu1 and note that it works optimally in buffer 2.1 or CS buffer. Select the Stu1 enzyme and click digest.</p><p>In the next window click on View gel under the Main options header. Select the option to run a virtual 1%gel using a pBR322-BstN1 digest. After the results appear take a screenshot and paste it into a blank document.</p><p>After this, navigate back to the main page of the NEBcutter tool and select New custom digest. This time search for the BsrG1 enzyme and note that it performs optimally in buffer 2.1 or buffer 3.1. Select the enzyme and click digest.</p><p>Under the Main options header click on View gel and select the options to run a virtual 1%gel using a pBR322-BstN1 digest. Take a screenshot of the results and save this in a text document. Finally, repeat these steps using both Stu1 and BsrG1 together in a single digest.</p><p>Again, take a screenshot and save the resulting gel. Before starting the restriction enzyme digests first put your aliquots of lambda DNA, NEB buffer 2.1, Stu1 enzyme, and BsrG1 enzyme into an ice bucket, and take this to your work area. The tube labeled Stu1 one will contain the Stu1 digest.</p><p>The tube labeled BsrG1 will contain the BsrG1 digest. And the tube labeled S plus B will contain the digest with both of the enzymes. You will now use the appropriate micropipetters to set up the reactions listed in this table.</p><p>First, pipette 40 microliters of water into each tube. Then using a fresh pipette tip each time dispense five microliters of buffer 2.1 into each tube and pipette up and down to mix. Next, add four microliters of lambda DNA to each tube, and then pipette 0.5 microliters of each enzyme into the appropriate tubes.</p><p>Finally, add a sufficient amount of water to each tube to bring the total volume up to 50 microliters. Next, incubate the tubes at 37 degrees Celsius for 15 to 60 minutes in a water bath and return the buffers and enzymes to the freezer. To prepare an agarose gel to run the digest in a 250-milliliter flask mix one gram of agarose powder with 100 milliliters of TAE buffer.</p><p>Microwave the solution for approximately 90 seconds at full power taking care to prevent the solution from boiling over. Next, carefully remove the solution from the microwave using a glove or heat pad and place it to cool for about 15 minutes. While the agarose solution cools set up a casting tray.</p><p>When the agarose solution is cool add an appropriate amount of 10, 000X SYBR Safe gel stain to the solution. Add the comb which is used to make the wells of the gel to the casting tray of the gel electrophoresis apparatus. Pour the agarose gel solution into the casting tray and allow it to solidify.</p><p>Then pull the comb straight up to remove it from the gel. And remove the dams from the casting tray. Place the gel in the electrophoresis chamber with the wells positioned closest to the negative or black electrode and fill the chamber with 1x TAE buffer enough to completely cover the gel.</p><p>Remove your samples from the water bath. Then add 8.3 microliters of 6X loading dye to each sample. Dispense 10 microliters of the pBR322-BstN1 digest which contains DNA fragments of known size into the first well.</p><p>Then load 25 microliters of sample Stu1 into the second well. Place 25 microliters of sample B into the third well and finally load 25 microliters of sample SB into the fourth well. To run the gel plug the leads into the electrophoresis chamber and then into the power supply.</p><p>The red positive lead should be at the end farthest away from the wells and the black negative lead should be nearest to the wells. This is because the samples will run from negative to positive. Turn on the power supply and set the gel box to run at 170 volts for approximately 60 minutes.</p><p>After the run is complete turn off the power to the gel box and disconnect the electrodes from the tank. Then remove the gel from the electrophoresis chamber and observe it with a UV transilluminator. Finally, use a cell phone camera to take a picture of the gel from directly above and parallel to the gel surface.</p><p>Compare the gel you ran in the lab to your virtual enzymatic digest with Stu1. The virtual digest with Stu1 resulted in seven bands. Count the bands in the Stu1 lane of your gel.</p><p>Did your gel confirm this prediction? Now look at the results for your BsrG1 digest. The virtual digest with BsrG1 resulted in six bands.</p><p>Count the bands in the BsrG1 lane. Did your gel confirm this prediction? Finally, look at the virtual digest with Stu1 and BsrG1.</p><p>The virtual digest with both enzymes resulted in 12 bands. Count the bands in the corresponding lane of your gel. Did your gel confirm this prediction?</p><p>Does the DNA banding pattern look similar to the virtual digest?</p>

What is Meiosis?

Meiosis is the process by which diploid cells divide to produce haploid daughter cells. In humans, each diploid cell contains 46 chromosomes, half from ...

Meiosis I

Meiosis II

Meiosis vs. Mitosis

Crossing Over

Crossing over is the exchange of genetic information between homologous chromosomes during prophase I of meiosis I. Genetic recombination gives rise to ...

Nondisjunction

Nondisjunction is the failure of homologous chromosomes or sister chromatids to separate correctly and move to the opposite poles of the cells. This ...

Spermatogenesis

Spermatogenesis is the process by which haploid sperm cells are produced in the male testes. It starts with stem cells located close to the outer rim of ...

Oogenesis

In human women, oogenesis produces one mature egg cell or ovum for every precursor cell that enters meiosis. This process differs in two unique ways from ...

Fertilization

During fertilization, an egg and sperm cell fuse to create a new diploid structure. In humans, the process occurs once the egg has been released from the ...

Cleavage and Blastulation

After a large-single-celled zygote is produced via fertilization, the process of cleavage occurs while zygotes travel through the uterine tube. Cleavage ...

Gastrulation

Gastrulation establishes the three primary tissues of an embryo: the ectoderm, mesoderm, and endoderm. This developmental process relies on a series of ...

Neurulation

Neurulation is the embryological process which forms the precursors of the central nervous system and occurs after gastrulation has established the three ...

Cell Migration

Cell migration, the process by which cells move from one location to another, is essential for the proper development and viability of organisms ...

Determination

During embryogenesis, cells become progressively committed to different fates through a two-step process: specification followed by determination. ...

Use models to explain how DNA is passed from parents to offspring through the processes of meiosis and fertilization in sexual reproduction.

LS.Bio.6.1

Mutations

Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations ...

genome-size-and-the-evolution-of-new-genes

Genome Size and the Evolution of New Genes

types-of-genetic-transfer-between-organisms

Types of Genetic Transfer Between Organisms

Base-pairing and DNA Repair

The DNA Replication Fork

Lagging Strand Synthesis

The Replisome

Proofreading

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template ...

Replication in Prokaryotes

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the ...

Replication in Eukaryotes

In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before ...

Overview of DNA Repair

Base Excision Repair

Nucleotide Excision Repair

DNA Distortion and Damage
Cells are regularly exposed to mutagens&#8212;factors in the environment that can damage DNA and generate mutations. UV ...

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the ...

Fixing Double-strand Breaks

Homologous Recombination

Gene Conversion

Restarting Stalled Replication Forks

Construct an explanation to summarize how inheritable genetic variations may result from: new genetic combinations in meiosis, mutations during replication, or mutations caused by environmental factors.

LS.Bio.6.2

An Overview of Genetic Analysis

An organism&#8217;s physical traits, or phenotype, are a product of its genotype, which is the combination of alleles (gene variants) inherited from its ...

Genetic Crosses

To dissect genetic processes or create organisms with novel suites of traits, scientists can perform genetic crosses, or the purposeful mating of two ...

Punnett Squares

Monohybrid Crosses

Dihybrid Crosses

Trihybrid Crosses

Trihybrid Crosses
Some of Mendel&#8217;s crosses examined three pairs of contrasting characteristics. Such a cross is called a trihybrid cross. A ...

Law of Independent Assortment

Chi-square Analysis

The chi-square test is a statistical hypothesis test. It is used to check whether there is a significant difference between an expected value and an ...

Pedigree Analysis

Multiple Allele Traits

Incomplete Dominance

Gregor Mendel&#39;s work (1822 - 1884) was primarily focused on pea plants. Through his initial experiments, he determined that every gene in a diploid ...

Lethal Alleles

Agouti: A Lethal Allele
Lucien Cu&#233;not discovered lethal alleles in 1905 while studying the inheritance of coat color in mice. The agouti gene is ...

Polygenic Traits

background-and-environment-affect-phenotype

Background and Environment Affect Phenotype

Although the genetic makeup of an organism plays a major role in determining the phenotype, there are also several environmental factors, such as ...

Use mathematics and computational thinking to predict the variation and distribution of expressed traits based on: Mendelian inheritance, co-dominance, incomplete dominance, multiple alleles, and sex-linked inheritance.

LS.Bio.7.1

Overview

A pedigree is a diagram displaying a family&#x2019;s history of a trait. Analyzing pedigrees can reveal (1) whether a trait is dominant or ...

Probability Laws

Overview

The probability of inheriting a trait can be calculated using the sum and product rules. The sum rule is used to calculate the probability of ...

The Concept of Multiple Allelism

Multiple allelism describes genes that exist in three or more allelic forms. Although diploid organisms, like humans, ...

Analyze and interpret data to explain how polygenic traits result in a wide range of phenotypes.

LS.Bio.7.2

Pleiotropy

Pleiotropy is the phenomenon in which a single gene impacts multiple, seemingly unrelated phenotypic traits. For example, defects in the SOX10 gene cause ...

Nature and Nurture

Many human characteristics, like height, are shaped by both nature&mdash;in other words, by our genes&mdash;and by nurture, or our environment. For ...

Construct an explanation to summarize how traits result from interactions of genetic factors (multiple genes and/or alleles) and environmental factors.

LS.Bio.7.3

PCR: The Polymerase Chain Reaction

The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling &#8211; cyles of temperature changes at fixed time ...

DNA Gel Electrophoresis

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of ...

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further ...

Restriction Enzymes

Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called ...

DNA Agarose Gel Electrophoresis

Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify ...

Analyze and interpret data to compare DNA samples.

Transgenic Organisms

Reproductive Cloning

CRISPR

Synthetic Biology

Synthetic biology is an interdisciplinary science that involves using principles from disciplines such as engineering, molecular biology, cell biology, ...

Ribosome Profiling

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences ...

Transgenic Plants

Recombinant DNA technology called transgenesis is often used to add a foreign gene or remove a detrimental gene from an organism. Such genetically ...

Gene Therapy

biofuels-producing-ethanol-from-cellulosic-material

Biofuels: Producing Ethanol from Cellulosic Material

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In this experiment, cellulosic material (such as corn stalks, leaves, ...

Testing For Genetically Modified Foods

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Genetic modification of foods has been a controversial issue due to debated ...

Obtain and communicate information that summarizes the impact of biotechnology applications on the individual, society, and the environment, including agriculture and medicine.

LS.Bio.8.2

Understand genetic mechanisms for variation.

DNA Isolation and Restriction Enzyme Analysis

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