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<h2 class="hd hd-2 unit-title">Reference Summary</h2>
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<h3><b>Big Picture: Cloning</b></h3>
<p><b>Cloning</b> is the process of isolating a gene of interest (GOI) and expressing it in an organism.</p>
<p>A <b>vector</b> is any carrier of DNA that can deliver that DNA, i.e. the GOI, to the cell and be replicated. One common example of a vector is a <b>plasmid</b>, which is a piece of small, circular DNA.</p>
<p> The cloning process is depicted in detail below.</p>
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<br /><br /><p> </p>
<h3><b> Restriction Endonucleases: Compatible Ends</b></h3>
<p>Restriction endonucleases, discussed in the previous section, are used to cut out and isolate the GOI (step 3 in the diagram above) and to cut the vector so that the GOI can be integrated into the vector (step 4 in the diagram above).<br /> In order for the GOI to integrate into the vector, the two need to be cut in a way that gives them compatible ends. In this way, they can match up and ligate into a new circular plasmid without any breaks. The different types of compatible ends are detailed below.</p>
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<p> Note that the GOI and the vector do not need to be cut with the same enzyme to be compatible. In the case of blunt ends, all blunt ends can match with other blunt ends. In the case of sticky ends, only the overhanging base pairs need to match up. <br />However, ligating DNAs from two different restriction sites may prevent the ligation point from serving as a restriction site in the future because you alter the sequence on the other half of the restriction site (in the case of blunt ends) or around the overhanging base pairs (in the case of sticky ends). </p>
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<h2 class="hd hd-2 unit-title">Exercise 1 (Blunt Ends)</h2>
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<p>The following questions concern the two restriction endonucleases shown here.</p>
<img src='/assets/courseware/v1/36d033a70b248180c53cec6e787f50e0/asset-v1:OCW+Pre-7.01+1T2020+type@asset+block/v1.png' summary="This image shows the sequences that are recongized by two restriction endonucleases (A l u 1 and D r a 1). Each recognition site is shown as the sequences of two complementary strands of D N A. There are slashes in the sequences to show where each D N A strand is being broken. There are dashes at the peripheries of each sequence to indicate that the sequence extends in both directions but that the identity of the sequence in those regions is irrelevant. For the A l u 1 recognition site, the top strand is written as 5 prime dash dash A G slash C T dash dash 3 prime and the bottom strand is written as 3 prime dash dash T C slash G A dash dash 5 prime. For the D r a 1 recognition site, the top strand is written as 5 prime dash T T T slash A A A dash dash 3 prime and the bottom strand is written as 3 prime dash dash A A A slash T T T dash dash 5 prime.">
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<p>Will the DNA fragments that result from digests with AluI and DraI ligate?<br/>Select the best answer.</p>
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<p>The ligated AluI/DraI fragments can subsequently cut with ___________.</p>
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Check all responses that accurately complete the phrase above.</p>
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<h2 class="hd hd-2 unit-title">Exercise 2 (Complementary Ends)</h2>
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<p>The following questions concern the restriction endonucleases shown here.</p>
<p><center><font size="+3"><strong> BglII </strong></font></center></p>
<p><center><font size="+3"><strong> 5'--A/GATCT--3' </strong></font></center></p>
<p><center><font size="+3"><strong> 3'--TCTAG/A--5' </strong></font></center></p>
<p><center><font size="+3"><strong> BamHI </strong></font></center></p>
<p><center><font size="+3"><strong> 5'--G/GATCC--3' </strong></font></center></p>
<p><center><font size="+3"><strong> 3'--CCTAG/G--5' </strong></font></center></p>
<p><center><font size="+3"><strong> SacI </strong></font></center></p>
<p><center><font size="+3"><strong> 5'--GAGCT/C--3' </strong></font></center></p>
<p><center><font size="+3"><strong> 3'--C/TCGAG--5' </strong></font></center></p>
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BglII
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<p>Which restriction sites give complementary ends to BglII? Select all that apply.</p>
<p>
(It may help to write out the products of cutting and then look for complementarity (base-pairing).)
</p>
<div class="wrapper-problem-response" tabindex="-1" aria-label="Question 1" role="group"><div class="choicegroup capa_inputtype" id="inputtype_69f419c1754941a1b15f3739bba913f1_2_1">
<fieldset aria-describedby="status_69f419c1754941a1b15f3739bba913f1_2_1">
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</label>
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BamHI
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<p>Which restriction sites give complementary ends to BamHI? Select all that apply.</p>
<p>
(It may help to write out the products of cutting and then look for complementarity (base-pairing).)
</p>
<div class="wrapper-problem-response" tabindex="-1" aria-label="Question 1" role="group"><div class="choicegroup capa_inputtype" id="inputtype_f3b939f425db46ad889f752789decffd_2_1">
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<input type="checkbox" name="input_f3b939f425db46ad889f752789decffd_2_1[]" id="input_f3b939f425db46ad889f752789decffd_2_1_choice_1" class="field-input input-checkbox" value="choice_1"/><label id="f3b939f425db46ad889f752789decffd_2_1-choice_1-label" for="input_f3b939f425db46ad889f752789decffd_2_1_choice_1" class="response-label field-label label-inline" aria-describedby="status_f3b939f425db46ad889f752789decffd_2_1"> SacI
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SacI
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<div class="problem-progress" id="block-v1:OCW+Pre-7.01+1T2020+type@problem+block@d03bc1c7d1a9473a9a96a69faab075fc-problem-progress"></div>
<div class="problem">
<div>
<p>Which restriction sites give complementary ends to SacI? Select all that apply.</p>
<p>
(It may help to write out the products of cutting and then look for complementarity (base-pairing).)
</p>
<div class="wrapper-problem-response" tabindex="-1" aria-label="Question 1" role="group"><div class="choicegroup capa_inputtype" id="inputtype_d03bc1c7d1a9473a9a96a69faab075fc_2_1">
<fieldset aria-describedby="status_d03bc1c7d1a9473a9a96a69faab075fc_2_1">
<div class="field">
<input type="checkbox" name="input_d03bc1c7d1a9473a9a96a69faab075fc_2_1[]" id="input_d03bc1c7d1a9473a9a96a69faab075fc_2_1_choice_0" class="field-input input-checkbox" value="choice_0"/><label id="d03bc1c7d1a9473a9a96a69faab075fc_2_1-choice_0-label" for="input_d03bc1c7d1a9473a9a96a69faab075fc_2_1_choice_0" class="response-label field-label label-inline" aria-describedby="status_d03bc1c7d1a9473a9a96a69faab075fc_2_1"> BglII
</label>
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<input type="checkbox" name="input_d03bc1c7d1a9473a9a96a69faab075fc_2_1[]" id="input_d03bc1c7d1a9473a9a96a69faab075fc_2_1_choice_1" class="field-input input-checkbox" value="choice_1"/><label id="d03bc1c7d1a9473a9a96a69faab075fc_2_1-choice_1-label" for="input_d03bc1c7d1a9473a9a96a69faab075fc_2_1_choice_1" class="response-label field-label label-inline" aria-describedby="status_d03bc1c7d1a9473a9a96a69faab075fc_2_1"> BamHI
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<h2 class="hd hd-2 unit-title">Exercise 3 (Ligating DNA fragments)</h2>
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<p>The following questions concern the restriction endonucleases shown here.</p>
<img src='/assets/courseware/v1/7b5f5397797b2a182441f3bfef0af422/asset-v1:OCW+Pre-7.01+1T2020+type@asset+block/v3.png' alt="This image shows the sequences that are recognized by two restriction endonucleases (N s i 1 and P s t 1). Each recognition site is shown as the sequences of two complementary strands of D N A. There are slashes in the sequences to show where each D N A strand is being broken. For the N s i 1 recognition site, the top strand is written as 5 prime A T G C A slash T 3 prime and the bottom strand is written as 3 prime T slash A C G T A 5 prime. For the P s t 1 recognition site, the top strand is written as 5 prime C T G C A slash G 3 prime and the bottom strand is written as 3 prime G slash A C G T C 5 prime.">
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Question 1
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<legend id="de2d35bbe6b34260a42dde1ccbbc8af9_2_1-legend" class="response-fieldset-legend field-group-hd">Will DNA fragments resulting from digests with NsiI and PstI ligate?<br/>Select the best answer.</legend>
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Question 2
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<p>If the answer to Question 1 is <b>yes</b>, type the nucleotides to show the resulting <b>6bp sequence</b> after ligation. (Type only letters with <b>no spaces</b>. Pay attention to the polarity denoted for each answer box, as you do not need to type the polarity in your answer.)</p>
<p>If the answer to Question 1 is <b>no</b>, type N/A in both answer boxes.</p>
<div class="wrapper-problem-response" tabindex="-1" aria-label="Question 1" role="group"><p><b>5' to 3'</b> (top strand):</p>
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<div class="wrapper-problem-response" tabindex="-1" aria-label="Question 2" role="group"><p><b>3' to 5'</b> (bottom strand):</p>
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Question 3
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<p>The 6 bp sequence after ligation will restore___________.</p>
<p>
Check all responses that accurately complete the phrase above.<br/>(Select only "N/A" if the answer to Quetsion 1 is <b>no</b>)</p>
<div class="wrapper-problem-response" tabindex="-1" aria-label="Question 1" role="group"><div class="choicegroup capa_inputtype" id="inputtype_85580e03617c4949b801d80fc2ef80aa_2_1">
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<input type="checkbox" name="input_85580e03617c4949b801d80fc2ef80aa_2_1[]" id="input_85580e03617c4949b801d80fc2ef80aa_2_1_choice_0" class="field-input input-checkbox" value="choice_0"/><label id="85580e03617c4949b801d80fc2ef80aa_2_1-choice_0-label" for="input_85580e03617c4949b801d80fc2ef80aa_2_1_choice_0" class="response-label field-label label-inline" aria-describedby="status_85580e03617c4949b801d80fc2ef80aa_2_1"> a PstI cut site
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</div>
<div class="field">
<input type="checkbox" name="input_85580e03617c4949b801d80fc2ef80aa_2_1[]" id="input_85580e03617c4949b801d80fc2ef80aa_2_1_choice_2" class="field-input input-checkbox" value="choice_2"/><label id="85580e03617c4949b801d80fc2ef80aa_2_1-choice_2-label" for="input_85580e03617c4949b801d80fc2ef80aa_2_1_choice_2" class="response-label field-label label-inline" aria-describedby="status_85580e03617c4949b801d80fc2ef80aa_2_1"> Neither
</label>
</div>
<div class="field">
<input type="checkbox" name="input_85580e03617c4949b801d80fc2ef80aa_2_1[]" id="input_85580e03617c4949b801d80fc2ef80aa_2_1_choice_3" class="field-input input-checkbox" value="choice_3"/><label id="85580e03617c4949b801d80fc2ef80aa_2_1-choice_3-label" for="input_85580e03617c4949b801d80fc2ef80aa_2_1_choice_3" class="response-label field-label label-inline" aria-describedby="status_85580e03617c4949b801d80fc2ef80aa_2_1"> N/A
</label>
</div>
<span id="answer_85580e03617c4949b801d80fc2ef80aa_2_1"/>
</fieldset>
<div class="indicator-container">
<span class="status unanswered" id="status_85580e03617c4949b801d80fc2ef80aa_2_1" data-tooltip="Not yet answered.">
<span class="sr">unanswered</span><span class="status-icon" aria-hidden="true"/>
</span>
</div>
</div></div>
<p>Note: Make sure you select all correct options!</p>
<div class="solution-span">
<span id="solution_85580e03617c4949b801d80fc2ef80aa_solution_1"/>
</div></div>
<div class="action">
<input type="hidden" name="problem_id" value="Question 3" />
<div class="submit-attempt-container">
<button type="button" class="submit btn-brand" data-submitting="Submitting" data-value="Submit" data-should-enable-submit-button="True" aria-describedby="submission_feedback_85580e03617c4949b801d80fc2ef80aa" >
<span class="submit-label">Submit</span>
</button>
<div class="submission-feedback" id="submission_feedback_85580e03617c4949b801d80fc2ef80aa">
<span class="sr">Some problems have options such as save, reset, hints, or show answer. These options follow the Submit button.</span>
</div>
</div>
<div class="problem-action-buttons-wrapper">
<span class="problem-action-button-wrapper">
<button type="button" class="reset problem-action-btn btn-default btn-small" data-value="Reset"><span class="icon fa fa-refresh" aria-hidden="true"></span><span aria-hidden="true">Reset</span><span class="sr">Reset your answer</span></button>
</span>
</div>
</div>
<div class="notification warning notification-gentle-alert
is-hidden"
tabindex="-1">
<span class="icon fa fa-exclamation-circle" aria-hidden="true"></span>
<span class="notification-message" aria-describedby="85580e03617c4949b801d80fc2ef80aa-problem-title">
</span>
<div class="notification-btn-wrapper">
<button type="button" class="btn btn-default btn-small notification-btn review-btn sr">Review</button>
</div>
</div>
<div class="notification warning notification-save
is-hidden"
tabindex="-1">
<span class="icon fa fa-save" aria-hidden="true"></span>
<span class="notification-message" aria-describedby="85580e03617c4949b801d80fc2ef80aa-problem-title">None
</span>
<div class="notification-btn-wrapper">
<button type="button" class="btn btn-default btn-small notification-btn review-btn sr">Review</button>
</div>
</div>
<div class="notification general notification-show-answer
is-hidden"
tabindex="-1">
<span class="icon fa fa-info-circle" aria-hidden="true"></span>
<span class="notification-message" aria-describedby="85580e03617c4949b801d80fc2ef80aa-problem-title">Answers are displayed within the problem
</span>
<div class="notification-btn-wrapper">
<button type="button" class="btn btn-default btn-small notification-btn review-btn sr">Review</button>
</div>
</div>
</div>
"
data-graded="False">
<p class="loading-spinner">
<i class="fa fa-spinner fa-pulse fa-2x fa-fw"></i>
<span class="sr">Loading…</span>
</p>
</div>
</div>
</div>
</div>
</div>