http://engineerbiology.org/w/index.php?title=20.109(F20):M2D1&feed=atom&action=history20.109(F20):M2D1 - Revision history2024-03-29T09:29:11ZRevision history for this page on the wikiMediaWiki 1.22.3http://engineerbiology.org/w/index.php?title=20.109(F20):M2D1&diff=48410&oldid=prevBecky Meyer: /* Part 4: Confirmation digest of pET-28b(+)_PF3D7_1351100 */2021-10-15T18:52:11Z<p><span dir="auto"><span class="autocomment">Part 4: Confirmation digest of pET-28b(+)_PF3D7_1351100</span></span></p>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#*Single digests of pET-28b(+)_PF3D7_1351100 (each enzyme used alone in a digest with pET-28b(+)_PF3D7_1351100).</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#*Single digests of pET-28b(+)_PF3D7_1351100 (each enzyme used alone in a digest with pET-28b(+)_PF3D7_1351100).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#Use the table below to calculate the volumes of each reagent that should be included in the confirmation digest reactions.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#Use the table below to calculate the volumes of each reagent that should be included in the confirmation digest reactions.</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div><del class="diffchange diffchange-inline">#</del>*The 20.109 enzyme stocks are always the "S" size and concentration when you search for them on the NEB website.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">Keep the following in mind as you consider which enzymes to use:</ins></div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div><del class="diffchange diffchange-inline">#</del>*To find the concentration of the enzyme(s) you choose, search the [<del class="diffchange diffchange-inline">http</del>://www.neb.com/products/restriction-endonucleases/restriction-endonucleases NEB site].</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">*Each enzyme should be present in 10 U quantity per reaction. As an example, the XbaI vial contains 20,000 U/mL, or 20 U/&mu;L.  </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>*The 20.109 enzyme stocks are always the "S" size and concentration when you search for them on the NEB website.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">*Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>*To find the concentration of the enzyme(s) you choose, search the [<ins class="diffchange diffchange-inline">https</ins>://www.neb.com/products/restriction-endonucleases/restriction-endonucleases NEB site].</div></td></tr>
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</table>Becky Meyerhttp://engineerbiology.org/w/index.php?title=20.109(F20):M2D1&diff=45775&oldid=prevBecky Meyer: /* Part 3: Ligation of PF3D7_1351100 insert and pET-28b(+) expression vector */2020-09-29T17:25:45Z<p><span dir="auto"><span class="autocomment">Part 3: Ligation of PF3D7_1351100 insert and pET-28b(+) expression vector</span></span></p>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Before you prepare a ligation, one very important step is to calculate the amounts of DNA that will be used in the reaction.  Ideally, you should use a 3:1 molar ratio of insert to vector (note: it is a molar ratio, not a volumetric ratio!). You will use the steps below to calculate the volume amount (based on the molar ratio!) of the PF3D7_1351100 insert and pET-28b(+) expression vector you would use to complete this ligation in the laboratory.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Before you prepare a ligation, one very important step is to calculate the amounts of DNA that will be used in the reaction.  Ideally, you should use a 3:1 molar ratio of insert to vector (note: it is a molar ratio, not a volumetric ratio!). You will use the steps below to calculate the volume amount (based on the molar ratio!) of the PF3D7_1351100 insert and pET-28b(+) expression vector you would use to complete this ligation in the laboratory.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>[[Image:Sp20 M1D1 recovery gel.png|thumb|right|300px|'''Recovery gel for ligation calculations.''' Lane 1 = <del class="diffchange diffchange-inline">PF3D7_1351100 insert</del>, Lane 2 = molecular weight ladder, and Lane 3 = <del class="diffchange diffchange-inline">pET-28b(+) expression vector</del>.]]</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>[[Image:Sp20 M1D1 recovery gel.png|thumb|right|300px|'''Recovery gel for ligation calculations.''' Lane 1 = <ins class="diffchange diffchange-inline">pET-28b(+) expression vector</ins>, Lane 2 = molecular weight ladder, and Lane 3 = <ins class="diffchange diffchange-inline">PF3D7_1351100 insert</ins>.]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Use the following information to calculate the volume of insert and vector needed to prepare a ligation with a 3:1 molar ratio (insert:vector).</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Use the following information to calculate the volume of insert and vector needed to prepare a ligation with a 3:1 molar ratio (insert:vector).</div></td></tr>
</table>Becky Meyerhttp://engineerbiology.org/w/index.php?title=20.109(F20):M2D1&diff=45774&oldid=prevBecky Meyer: /* Introduction */2020-09-29T15:29:37Z<p><span dir="auto"><span class="autocomment">Introduction</span></span></p>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>==Introduction==</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>==Introduction==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Though the theme of Module <del class="diffchange diffchange-inline">1 </del>is focused on screening for small molecules that bind the PF3D7_1351100 protein, today will focus on a few key techniques used in DNA engineering.  Because the sequence of proteins is determined by the sequence of the genes that encode them, learning how to manipulate DNA is an important first step.  Today you will complete the cloning steps used to incorporate the gene that encodes the PF3D7_1351100 protein into an expression vector.  The expression vector contains the genetic elements needed to express and purify a protein of interest.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Though the theme of Module <ins class="diffchange diffchange-inline">2 </ins>is focused on screening for small molecules that bind the PF3D7_1351100 protein, today will focus on a few key techniques used in DNA engineering.  Because the sequence of proteins is determined by the sequence of the genes that encode them, learning how to manipulate DNA is an important first step.  Today you will complete the cloning steps used to incorporate the gene that encodes the PF3D7_1351100 protein into an expression vector.  The expression vector contains the genetic elements needed to express and purify a protein of interest.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Expression vectors contain several features important for cloning, plasmid replication, and protein expression -- all of which are important for purifying high-quality protein.  To generate this expression plasmid, two common DNA engineering techniques were used: restriction enzyme digestion and ligation.  First, the PF3D7 insert was synthesized such that the gene sequence is flanked by restriction enzymes sites.  Next, this fragment and the vector were digested to create compatible ends.  Last, the compatible ends of the digested insert and vector were  ligated to generate the pET-28b(+)_PF3D7_1351100 expression plasmid.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Expression vectors contain several features important for cloning, plasmid replication, and protein expression -- all of which are important for purifying high-quality protein.  To generate this expression plasmid, two common DNA engineering techniques were used: restriction enzyme digestion and ligation.  First, the PF3D7 insert was synthesized such that the gene sequence is flanked by restriction enzymes sites.  Next, this fragment and the vector were digested to create compatible ends.  Last, the compatible ends of the digested insert and vector were  ligated to generate the pET-28b(+)_PF3D7_1351100 expression plasmid.</div></td></tr>
</table>Becky Meyerhttp://engineerbiology.org/w/index.php?title=20.109(F20):M2D1&diff=45773&oldid=prevBecky Meyer: /* Part 1: Synthesis and restriction enzyme digest of PF3D7_1351100 insert */2020-09-28T19:52:44Z<p><span dir="auto"><span class="autocomment">Part 1: Synthesis and restriction enzyme digest of PF3D7_1351100 insert</span></span></p>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Part 1: Synthesis and restriction enzyme digest of PF3D7_1351100 insert===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Part 1: Synthesis and restriction enzyme digest of PF3D7_1351100 insert===</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">To use SnapGene software off campus you must log into a VPN connection prior to opening the SnapGene. Here is the link to the [https://ist.mit.edu/cisco-anyconnect VPN download] and [http://kb.mit.edu/confluence/x/6QPn installation instructions]. Also you will need to update the SnapGene license number if you have not opened the application since March. The new license information can be found [http://downloads.mit.edu/released/snapgene/group-name_registration-code.txt here]. </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>As discussed in the [[20.109(F20):Module 2|M2 project overview]], PF3D7_1351100 is an essential protein of unknown function in ''Plasmodium falciparum''.  In an effort to study PF3D7_1351100 a researcher in the Niles Laboratory, Dr. Khan Osman, cloned the gene that encodes the protein into an expression plasmid.  Rather than amplifying the gene from the ''P. falciparum'' genome, gBlock synthesis technology was used.  A gBlock is a double-stranded DNA segment that is synthesized commercially without the use of live cells.  To generate a gBlock, a researcher simply submits the basepair sequence to be synthesized.  This method is useful for several reasons including: 1. it can be technically difficult to amplify genes from certain organisms, and 2. it can be easier to modify DNA that is synthetically generated.  For this project, the PF3D7_1351100 gBlock sequence was modified such that the codon usage was optimized for expression in ''E. coli'' cells.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>As discussed in the [[20.109(F20):Module 2|M2 project overview]], PF3D7_1351100 is an essential protein of unknown function in ''Plasmodium falciparum''.  In an effort to study PF3D7_1351100 a researcher in the Niles Laboratory, Dr. Khan Osman, cloned the gene that encodes the protein into an expression plasmid.  Rather than amplifying the gene from the ''P. falciparum'' genome, gBlock synthesis technology was used.  A gBlock is a double-stranded DNA segment that is synthesized commercially without the use of live cells.  To generate a gBlock, a researcher simply submits the basepair sequence to be synthesized.  This method is useful for several reasons including: 1. it can be technically difficult to amplify genes from certain organisms, and 2. it can be easier to modify DNA that is synthetically generated.  For this project, the PF3D7_1351100 gBlock sequence was modified such that the codon usage was optimized for expression in ''E. coli'' cells.</div></td></tr>
</table>Becky Meyerhttp://engineerbiology.org/w/index.php?title=20.109(F20):M2D1&diff=45763&oldid=prevBecky Meyer: /* Part 1: Synthesis and restriction enzyme digest of PF3D7_1351100 insert */2020-09-25T15:17:07Z<p><span dir="auto"><span class="autocomment">Part 1: Synthesis and restriction enzyme digest of PF3D7_1351100 insert</span></span></p>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Part 1: Synthesis and restriction enzyme digest of PF3D7_1351100 insert===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Part 1: Synthesis and restriction enzyme digest of PF3D7_1351100 insert===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>As discussed in the [[20.109(F20):Module 2|<del class="diffchange diffchange-inline">M1 </del>project overview]], PF3D7_1351100 is an essential protein of unknown function in ''Plasmodium falciparum''.  In an effort to study PF3D7_1351100 a researcher in the Niles Laboratory, Dr. Khan Osman, cloned the gene that encodes the protein into an expression plasmid.  Rather than amplifying the gene from the ''P. falciparum'' genome, gBlock synthesis technology was used.  A gBlock is a double-stranded DNA segment that is synthesized commercially without the use of live cells.  To generate a gBlock, a researcher simply submits the basepair sequence to be synthesized.  This method is useful for several reasons including: 1. it can be technically difficult to amplify genes from certain organisms, and 2. it can be easier to modify DNA that is synthetically generated.  For this project, the PF3D7_1351100 gBlock sequence was modified such that the codon usage was optimized for expression in ''E. coli'' cells.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>As discussed in the [[20.109(F20):Module 2|<ins class="diffchange diffchange-inline">M2 </ins>project overview]], PF3D7_1351100 is an essential protein of unknown function in ''Plasmodium falciparum''.  In an effort to study PF3D7_1351100 a researcher in the Niles Laboratory, Dr. Khan Osman, cloned the gene that encodes the protein into an expression plasmid.  Rather than amplifying the gene from the ''P. falciparum'' genome, gBlock synthesis technology was used.  A gBlock is a double-stranded DNA segment that is synthesized commercially without the use of live cells.  To generate a gBlock, a researcher simply submits the basepair sequence to be synthesized.  This method is useful for several reasons including: 1. it can be technically difficult to amplify genes from certain organisms, and 2. it can be easier to modify DNA that is synthetically generated.  For this project, the PF3D7_1351100 gBlock sequence was modified such that the codon usage was optimized for expression in ''E. coli'' cells.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>[[Image:Fa20 M2D1 insert synthesis and digest .png|thumb|right|300px|]]</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>[[Image:Fa20 M2D1 insert synthesis and digest .png|thumb|right|300px|]]</div></td></tr>
</table>Becky Meyerhttp://engineerbiology.org/w/index.php?title=20.109(F20):M2D1&diff=45669&oldid=prevNoreen Lyell: /* Part 3: Ligation of PF3D7_1351100 insert and pET-28b(+) expression vector */2020-09-02T17:46:33Z<p><span dir="auto"><span class="autocomment">Part 3: Ligation of PF3D7_1351100 insert and pET-28b(+) expression vector</span></span></p>
<table class='diff diff-contentalign-left'>
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr style='vertical-align: top;'>
<td colspan='2' style="background-color: white; color:black; text-align: center;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 17:46, 2 September 2020</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 118:</td>
<td colspan="2" class="diff-lineno">Line 118:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#*Remember, this number should be 3-fold more than the moles of vector to accomplish a 3:1 molar ratio.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#*Remember, this number should be 3-fold more than the moles of vector to accomplish a 3:1 molar ratio.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#Calculate the volume of insert that contains the appropriate moles of insert.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#Calculate the volume of insert that contains the appropriate moles of insert.</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>#One additional consideration is the volume of the reaction.  The total volume of the ligation reaction <del class="diffchange diffchange-inline">A 15 &mu;L scale ligation </del>should not be greater than 15 &mu;L.  In this, the total volume of the insert and vector should not be greater than 13.5 &mu;L as additional reagents are required in the reaction.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>#One additional consideration is the volume of the reaction.  The total volume of the ligation reaction should not be greater than 15 &mu;L.  In this, the total volume of the insert and vector should not be greater than 13.5 &mu;L as additional reagents are required in the reaction.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#*If the insert and vector volume total greater than 13.5 &mu;L, you should (1) scale down both DNA amounts, using less than 50 ng backbone and/or (2) stray from the ideal 3:1 molar ratio.  </div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#*If the insert and vector volume total greater than 13.5 &mu;L, you should (1) scale down both DNA amounts, using less than 50 ng backbone and/or (2) stray from the ideal 3:1 molar ratio.  </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#*You may ask the teaching faculty for advice during class if you are unsure what choice is best.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#*You may ask the teaching faculty for advice during class if you are unsure what choice is best.</div></td></tr>
</table>Noreen Lyellhttp://engineerbiology.org/w/index.php?title=20.109(F20):M2D1&diff=45668&oldid=prevNoreen Lyell: /* Introduction */2020-09-02T17:45:45Z<p><span dir="auto"><span class="autocomment">Introduction</span></span></p>
<table class='diff diff-contentalign-left'>
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr style='vertical-align: top;'>
<td colspan='2' style="background-color: white; color:black; text-align: center;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 17:45, 2 September 2020</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 6:</td>
<td colspan="2" class="diff-lineno">Line 6:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>==Introduction==</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>==Introduction==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Though the theme of Module 1 is focused on screening for small molecules that bind the PF3D7_1351100 protein, today will focus on a few key techniques used in DNA engineering.  Because the sequence of proteins is determined by the sequence of the genes that encode them, learning how to manipulate DNA is an important first step.  Today you will complete the cloning steps used to incorporate the gene that encodes the PF3D7_1351100 protein into an expression vector.  The expression vector contains the genetic elements needed to express and purify <del class="diffchange diffchange-inline">of </del>a protein of interest.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Though the theme of Module 1 is focused on screening for small molecules that bind the PF3D7_1351100 protein, today will focus on a few key techniques used in DNA engineering.  Because the sequence of proteins is determined by the sequence of the genes that encode them, learning how to manipulate DNA is an important first step.  Today you will complete the cloning steps used to incorporate the gene that encodes the PF3D7_1351100 protein into an expression vector.  The expression vector contains the genetic elements needed to express and purify a protein of interest.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Expression vectors contain several features important for cloning, plasmid replication, and protein expression -- all of which are important for purifying high-quality protein.  To generate this expression plasmid, two common DNA engineering techniques were used: restriction enzyme digestion and ligation.  First, the PF3D7 insert was synthesized such that the gene sequence is flanked by restriction enzymes sites.  Next, this fragment and the vector were digested to create compatible ends.  Last, the compatible ends of the digested insert and vector were  ligated to generate the pET-28b(+)_PF3D7_1351100 expression plasmid.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Expression vectors contain several features important for cloning, plasmid replication, and protein expression -- all of which are important for purifying high-quality protein.  To generate this expression plasmid, two common DNA engineering techniques were used: restriction enzyme digestion and ligation.  First, the PF3D7 insert was synthesized such that the gene sequence is flanked by restriction enzymes sites.  Next, this fragment and the vector were digested to create compatible ends.  Last, the compatible ends of the digested insert and vector were  ligated to generate the pET-28b(+)_PF3D7_1351100 expression plasmid.</div></td></tr>
</table>Noreen Lyellhttp://engineerbiology.org/w/index.php?title=20.109(F20):M2D1&diff=45667&oldid=prevNoreen Lyell: /* Introduction */2020-09-02T17:45:23Z<p><span dir="auto"><span class="autocomment">Introduction</span></span></p>
<table class='diff diff-contentalign-left'>
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr style='vertical-align: top;'>
<td colspan='2' style="background-color: white; color:black; text-align: center;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 17:45, 2 September 2020</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 8:</td>
<td colspan="2" class="diff-lineno">Line 8:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Though the theme of Module 1 is focused on screening for small molecules that bind the PF3D7_1351100 protein, today will focus on a few key techniques used in DNA engineering.  Because the sequence of proteins is determined by the sequence of the genes that encode them, learning how to manipulate DNA is an important first step.  Today you will complete the cloning steps used to incorporate the gene that encodes the PF3D7_1351100 protein into an expression vector.  The expression vector contains the genetic elements needed to express and purify of a protein of interest.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Though the theme of Module 1 is focused on screening for small molecules that bind the PF3D7_1351100 protein, today will focus on a few key techniques used in DNA engineering.  Because the sequence of proteins is determined by the sequence of the genes that encode them, learning how to manipulate DNA is an important first step.  Today you will complete the cloning steps used to incorporate the gene that encodes the PF3D7_1351100 protein into an expression vector.  The expression vector contains the genetic elements needed to express and purify of a protein of interest.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Expression vectors contain several features important <del class="diffchange diffchange-inline">for </del>for cloning, plasmid replication, and protein expression -- all of which are important for purifying high-quality protein.  To generate this expression plasmid, two common DNA engineering techniques were used: restriction enzyme digestion and ligation.  First, the PF3D7 insert was synthesized such that the gene sequence is flanked by restriction enzymes sites.  Next, this fragment and the vector were digested to create compatible ends.  Last, the compatible ends of the digested insert and vector were  ligated to generate the pET-28b(+)_PF3D7_1351100 expression plasmid.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Expression vectors contain several features important for cloning, plasmid replication, and protein expression -- all of which are important for purifying high-quality protein.  To generate this expression plasmid, two common DNA engineering techniques were used: restriction enzyme digestion and ligation.  First, the PF3D7 insert was synthesized such that the gene sequence is flanked by restriction enzymes sites.  Next, this fragment and the vector were digested to create compatible ends.  Last, the compatible ends of the digested insert and vector were  ligated to generate the pET-28b(+)_PF3D7_1351100 expression plasmid.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>[[Image:Fa20 M2D1 cloning schematic.png|thumb|center|450px|'''Schematic of pET-28b(+)_PF3D7_1351100 cloning.''']]</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>[[Image:Fa20 M2D1 cloning schematic.png|thumb|center|450px|'''Schematic of pET-28b(+)_PF3D7_1351100 cloning.''']]</div></td></tr>
</table>Noreen Lyellhttp://engineerbiology.org/w/index.php?title=20.109(F20):M2D1&diff=45610&oldid=prevBecky Meyer: /* Part 5: Electrophorese confirmation digests */2020-08-18T22:53:51Z<p><span dir="auto"><span class="autocomment">Part 5: Electrophorese confirmation digests</span></span></p>
<table class='diff diff-contentalign-left'>
<col class='diff-marker' />
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<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 22:53, 18 August 2020</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>For the digests that were prepared in the previous laboratory session, a 1% agarose gel with SYBR Safe DNA stain was used to separate the DNA fragments in the four digest reactions.  In addition, a well was loaded with a molecular weight marker (also called a DNA ladder) to determine the size of the fragments.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>For the digests that were prepared in the previous laboratory session, a 1% agarose gel with SYBR Safe DNA stain was used to separate the DNA fragments in the four digest reactions.  In addition, a well was loaded with a molecular weight marker (also called a DNA ladder) to determine the size of the fragments.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div><font color = #0d368e>'''To ensure the steps included below are clear, please watch the video tutorial <del class="diffchange diffchange-inline">(</del>linked [[https://www.dropbox.com/s/ijo0d5pi8pg2f5c/DNA%20Gel%20electrophoresis.mp4?dl=0 <del class="diffchange diffchange-inline">| here</del>]]<del class="diffchange diffchange-inline">)</del>.  The steps are detailed below so you can follow along!'''</font color></div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><font color = #0d368e>'''To ensure the steps included below are clear, please watch the video tutorial linked <ins class="diffchange diffchange-inline">here: </ins>[[https://www.dropbox.com/s/ijo0d5pi8pg2f5c/DNA%20Gel%20electrophoresis.mp4?dl=0 <ins class="diffchange diffchange-inline">DNA gel electrophoresis</ins>]].  The steps are detailed below so you can follow along!'''</font color></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#Add 5 &mu;L of 6x loading dye to the digests.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#Add 5 &mu;L of 6x loading dye to the digests.</div></td></tr>
</table>Becky Meyerhttp://engineerbiology.org/w/index.php?title=20.109(F20):M2D1&diff=45609&oldid=prevBecky Meyer: /* Part 5: Electrophorese confirmation digests */2020-08-18T22:53:11Z<p><span dir="auto"><span class="autocomment">Part 5: Electrophorese confirmation digests</span></span></p>
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<td colspan='2' style="background-color: white; color:black; text-align: center;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 22:53, 18 August 2020</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>For the digests that were prepared in the previous laboratory session, a 1% agarose gel with SYBR Safe DNA stain was used to separate the DNA fragments in the four digest reactions.  In addition, a well was loaded with a molecular weight marker (also called a DNA ladder) to determine the size of the fragments.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>For the digests that were prepared in the previous laboratory session, a 1% agarose gel with SYBR Safe DNA stain was used to separate the DNA fragments in the four digest reactions.  In addition, a well was loaded with a molecular weight marker (also called a DNA ladder) to determine the size of the fragments.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div><font color = #0d368e>'''To ensure the steps included below are clear, please watch the video tutorial (linked [[ | here]]).  The steps are detailed below so you can follow along!'''</font color></div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><font color = #0d368e>'''To ensure the steps included below are clear, please watch the video tutorial (linked [[<ins class="diffchange diffchange-inline">https://www.dropbox.com/s/ijo0d5pi8pg2f5c/DNA%20Gel%20electrophoresis.mp4?dl=0 </ins>| here]]).  The steps are detailed below so you can follow along!'''</font color></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#Add 5 &mu;L of 6x loading dye to the digests.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#Add 5 &mu;L of 6x loading dye to the digests.</div></td></tr>
</table>Becky Meyer