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<!-- header begins --> | <!-- header begins --> | ||
<div id="header"> | <div id="header"> | ||
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<p><img src="https://static.igem.org/mediawiki/igem.org/5/5d/Wiki_Wide_Band.jpg" alt="logo_HKU" width="859" height="160" /></p> | <p><img src="https://static.igem.org/mediawiki/igem.org/5/5d/Wiki_Wide_Band.jpg" alt="logo_HKU" width="859" height="160" /></p> | ||
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</ul> | </ul> | ||
<p> </p> | <p> </p> | ||
| - | <p style="text-align: justify"> </p> | + | <p style="text-align: justify"> </p><h2 style="font-variant: normal; vertical-align: baseline; clear: left; font-family: Gentium Basic; letter-spacing: normal; orphans: 2; text-align: justify; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; padding: 0px; margin-left:0px; margin-right:0px"><span style="font-weight: 400"><font face="Trebuchet MS" size="6"><u>Molecular Cloning Protocols</font></u></span></h2> |
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<div align="left"> | <div align="left"> | ||
| - | + | <table id="toc" class="toc"> | |
| + | <tr><td height="224"><div id="toctitl"><h2><u> | ||
| + | <font face="Tahoma" color="#008000" size="2">Contents<br> | ||
| + | </font></u><font face="Trebuchet MS" style="font-size: 8pt" color="#000000"> | ||
| + | <a href="#Transforming_Competent_E.coli_with_the_Plasmid:_">1.1 Transforming Competent E.coli with the Plasmid</a><br> | ||
| + | <a href="#Miniprep_to_Extract_the_Plasmid_from_E.coli_(QIAprep_Spin_Miniprep_Kit):__">1.2 Miniprep to Extract the Plasmid from E.coli | ||
| + | </a> <br> | ||
| + | <a href="#Midiprep_for_Large_Volumes_of_Culture_(QIAGEN_Plasmid_Midi_Kit):_">1.3 Midiprep for Large Volumes of Culture | ||
| + | </a> <br> | ||
| + | <a href="#PCR_Amplification_of_Gene_from_Bacterial_Genome/Standard_Biobrick_Parts_">1.4 PCR Amplification of Gene from Bacterial Genome | ||
| + | </a> <br> | ||
| + | <a href="#PCR_Clean-Up_–_Qiagen_QIAquick_PCR_Purification:_">1.5 PCR Cleanup Qiagen QIAquick PCR Purification</a><br> | ||
| + | <a href="#Colony_PCR_">1.5.1 Colony PCR</a><br> | ||
| + | <a href="#Agarose_Gel_Electrophoresis_">1.5.2 Agarose Gel Electrophoresis | ||
| + | </a> <br> | ||
| + | <a href="#Gel_Purification_of_DNA_(Qiagen_QIAquick_Gel_Extraction_Kit)">1.6 Gel Purification of DNA (Qiagen QIAquick Gel Extraction Kit)</a><br> | ||
| + | <a href="#Determining_DNA_Concentration_Using_NanoDrop_Spectrophotometry">1.7 Determining DNA Concentration using NanoDrop Spectrophotometry</a> <br> | ||
| + | <a href="#DNA_Digestion_">1.8 DNA digestion</a> <br> | ||
| + | <a href="#Dephosphorylation_of_5_Ends_of_Vector_Backbone">1.9 Dephosphorylation of 5' Ends of Vector Backbone</a> <br> | ||
| + | <a href="#Vector-Insert_Ligation">2.0 Vector-Insert Litigation</a></font></h2> | ||
| + | <p><font face="Trebuchet MS"><span style="font-size: 8pt"> | ||
| + | <a href="#PCR_Deletion_(Site-Directed_Mutagenesis)_Reaction">2.1 PCR | ||
| + | Deletion Reaction</a></span></font></div> | ||
| + | </td></tr></table></div> | ||
| + | <script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <p> </p> | ||
| + | <div align="left"> | ||
| + | <table border="0" width="94%" height="2668"> | ||
<tr> | <tr> | ||
| - | <td | + | <td height="2664"> |
| - | + | <p style="font-variant: normal; vertical-align: baseline; clear: left; color: #232323; font-family: Gentium Basic; letter-spacing: normal; orphans: 2; text-align: justify; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; padding: 0px; margin-left:0px; margin-right:0px"> | |
| - | + | <span style="font-family: Trebuchet MS; font-weight: 400; text-decoration: underline"> | |
| - | + | <i><font size="4" color="#000000" id="Transforming_Ecoli"> | |
| - | + | <a name="Transforming_Competent_E.coli_with_the_Plasmid:_"> | |
| - | + | <b> | |
| - | + | Transforming Competent E.coli with the | |
| - | + | Plasmid:</b> </a> <br> | |
| - | + | <br> | |
| - | + | </font></i></span><font color="#000000"></b><font size="2" face="Tahoma">We chose the biobrick | |
| - | + | K137076 to ligate to the pvdQ gene for the following reasons: </font></font> | |
| - | + | </font></p> | |
| - | + | <ul> | |
| - | + | <li><font face="Tahoma" size="2" color="#000000">Take out the competent E-coli | |
| - | + | cells from the -80 freezer. (Keep all tubes on ice).</font></li> | |
| - | + | <li><font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">Add 1uL | |
| - | + | of the plasmid DNA in 100uL competent cells (if from Kit). For | |
| - | + | transformation of ligated product, add 10uL of the ligated plasmid DNA in | |
| - | + | 100uL competent cells.</span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Incubate on ice for 10 min. </span></font></li> | |
| - | + | <li><font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">Place | |
| - | + | in water bath at 42°C for 90s.</span></font></li> | |
| - | + | <li><font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">Place | |
| - | + | immediately back on ice for at least 2 min.</span></font></li> | |
| - | + | <li><font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">Add | |
| - | + | 800uL LB </span></font><font size="2" color="#000000"> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400">broth. Incubate for 1hr | |
| - | + | at 37°C shaker</span></font><font color="#000000"><span style="font-size:11.0pt;font-family:"Times New Roman","serif"">.</span></font></li> | |
| - | + | <li><font size="2" color="#000000" face="Tahoma">Centrifuge at 130rpm for 5min | |
| - | + | to remove supernatant. Re-suspend the pellet in about 10uL of supernatant.</font></li> | |
| - | + | <li><font face="Tahoma" size="2" color="#000000">Spread the entire re-suspended | |
| - | + | pellet on ampicillin agar dishes.</font></li> | |
| - | + | <li><font face="Tahoma" size="2" color="#000000">Incubate for 12-16 hours at 37 | |
| - | + | </font><font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">°C.</span></font></li> | |
| - | + | </ul> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | |
| - | + | <font color="#000000"><b> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400; text-decoration: underline"> | |
| - | + | Notes:</span></b></font></p> | |
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<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
<font color="#000000"> | <font color="#000000"> | ||
<span style="font-size: 10pt; font-family: Tahoma; ">- </span> | <span style="font-size: 10pt; font-family: Tahoma; ">- </span> | ||
<span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| - | Colonies grown on plate were selected for colony PCR screening.</span></font>< | + | Colonies grown on plate were selected for colony PCR screening.</span></font><br> |
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<font color="#000000"> | <font color="#000000"> | ||
<span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">- | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">- | ||
Correct colonies were then cultured in 5 mL LB Broth with 0.5uL | Correct colonies were then cultured in 5 mL LB Broth with 0.5uL | ||
ampicillin for subsequent screening or mass culturing. </span> | ampicillin for subsequent screening or mass culturing. </span> | ||
| - | < | + | <br> |
| - | < | + | </font><font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">- |
| - | + | Mass culturing involved adding 200uL of the culture into 35mL LB Broth | |
| - | + | with 35uL ampicillin. </span></font></p> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left"><font color="#000000" size="4"><b> | |
| - | + | <u> | |
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | + | <span style="font-family: Trebuchet MS; "><i> |
| - | + | <a name="Miniprep_to_Extract_the_Plasmid_from_E.coli_(QIAprep_Spin_Miniprep_Kit):__"> | |
| - | + | Miniprep to Extract the Plasmid from E.coli (QIAprep Spin Miniprep Kit): | |
| - | + | </a> </i></span> | |
| - | + | </u></b></font></p> | |
| - | + | ||
| - | + | ||
| - | Extract the Plasmid from E.coli (QIAprep Spin Miniprep Kit): </i></span> | + | |
| - | + | ||
<ul> | <ul> | ||
| - | + | <li> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400"> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | Transfer some of the 5mL bacterial culture into a microcentrifuge | |
| - | + | tube. Pellet by centrifugation at 13,500 rpm for 1 min. Repeat till | |
| - | + | all the culture has been pelleted. </font></span></li> | |
| - | + | <li> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400"> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | Resuspend the pellet in 250uL Buffer P1. </font></span></li> | |
| - | + | <li> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400"> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | Add 250uL Buffer P2 (Lysis Buffer). Mix thoroughly by inverting the | |
| - | + | tube 4-6 times. Do not allow prolonged lysis.</font></span></li> | |
| - | + | <li> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400"> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | Immediately add 350uL Buffer N3 (Neutralization Buffer). Mix | |
| - | + | immediately by inverting the tube. </font></span></li> | |
| - | + | <li> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400"> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | Centrifuge for 10 minutes at 13,500 rpm. </font></span></li> | |
| - | + | <li> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400"> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | Apply the resulting supernatant to the QIAprep spin column by | |
| - | + | pipetting. Centrifuge for 1 minute at 13,500 rpm. </font></span> | |
| - | + | </li> | |
| - | + | <li> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400"> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | Wash the QIAprep spin column by applying 0.75mL Buffer PE. | |
| - | + | Centrifuge for 1 minute at 13,500 rpm.</font></span></li> | |
| - | + | <li> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400"> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | Centrifuge for an additional 1 minute to remove residual ethanol. | |
| - | + | </font></span></li> | |
| - | + | <li> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400"> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | Place the QIAprep spin column in microcentrifuge tube. Elute DNA by | |
| - | + | adding 50uL warm H<sub>2</sub>O. </font></span></li> | |
| - | + | <li> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400"> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | Centrifuge for 1 minute at 13,500 rpm.</font></span></li> | |
| - | + | </ul> | |
| - | + | <p style="text-autospace: none; text-align: left; "><font color="#000000"><u><span style="font-size: 14pt; font-family: Trebuchet MS; font-style: italic"> | |
| - | + | <a name="Midiprep_for_Large_Volumes_of_Culture_(QIAGEN_Plasmid_Midi_Kit):_">Midiprep for Large Volumes of Culture (QIAGEN Plasmid Midi Kit): | |
| - | + | </a> </span> | |
| - | + | </u></font></p> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left; "> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Refer to the QIAGEN website for the protocol. </span></font></p> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left; "> | |
| - | + | <font color="#000000"><u> | |
| - | <p style="text-autospace: none; text-align: left; | + | <span style="font-size: 14pt; font-family: Trebuchet MS; font-style: italic"> |
| - | + | <a name="PCR_Amplification_of_Gene_from_Bacterial_Genome/Standard_Biobrick_Parts_">PCR Amplification of Gene from Bacterial Genome/Standard Biobrick Parts | |
| - | + | </a> | |
| - | Midiprep for Large Volumes of Culture (QIAGEN Plasmid Midi Kit): </span> | + | </span></u></font></p> |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left; "><font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left; | + | 37.5μL ddH2O</span></font><br> |
| - | + | <font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 5.0μL 10x PCR Buffer</span></font><br> | |
| - | + | <font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left; | + | 2.5μL dNTPs</span></font><br> |
| - | + | <font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 1.0μL Forward Primer (Prefix)</span></font><br> | |
| - | + | <font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 1.0μL Reverse Primer (Suffix)</span></font><br> | |
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | + | <font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> |
| - | + | 1.0μL Template DNA<br> | |
| - | + | </span></font><font color="#000000"><u><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">1.0 | |
| - | + | μL RTaq DNA Polymerase</span></u></font><br> | |
| - | + | <font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 50.0μL Total</span></font> </p> | |
| - | + | <font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"><u>Note: </u><br> | |
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- For sequential PCR reactions: After the first PCR is complete, perform | - For sequential PCR reactions: After the first PCR is complete, perform | ||
PCR clean-up. Then use the product to conduct the second PCR reaction, | PCR clean-up. Then use the product to conduct the second PCR reaction, | ||
| - | after which gel purification needs to be carried out. </span>< | + | after which gel purification needs to be carried out. </span><br> |
| - | + | </font><font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | |
| - | + | </span></span></font><font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">The | |
| - | + | volume of DNA used in the second PCR reaction depends on the | |
| - | + | concentration of the DNA after the PCR Clean-up. </span></font> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left"><font color="#000000"><i><u><span style="font-size: 14pt; font-family: Trebuchet MS"> | |
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | + | <a name="PCR_Clean-Up_–_Qiagen_QIAquick_PCR_Purification:_">PCR Clean-Up – |
| - | + | Qiagen QIAquick PCR Purification: </a> </span></u></i></font> | |
| - | + | <ul> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | Qiagen QIAquick PCR Purification: </span></u></i></font | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> |
| - | + | Add 5 volumes of Buffer PB to 1 volume of PCR mix. </span></font> | |
| - | + | </li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Place this mix within the QIAquick column. Centrifuge at 13,500 rpm | |
| - | + | for 1 minute. </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Discard flow through and centrifuge again to allow all the sample to | |
| - | + | pass through.</span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Wash with 750uL Buffer PE. Centrifuge at 13,500 rpm for 1 minute. | |
| - | + | </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Discard flow through and centrifuge again to remove all residual | |
| - | + | buffer. </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Place the column in a micro centrifuge tube. </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Apply 50uL of pre-warmed distilled H2O to the column. Stand for at | |
| - | + | least 2 minutes.</span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Centrifuge at 13,500 rpm for 1 minute. </span></font></li> | |
| - | + | </ul> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left"><font color="#000000"><u> | |
| - | + | <a name="Colony_PCR_"><i><font face="Trebuchet MS"><span style="font-size: 10pt">Colony PCR</span></font></i></a></u><a name="Colony_PCR_"> | |
| - | + | </a> | |
| - | + | </font> | |
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| - | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | + | |
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| - | + | ||
| - | + | ||
| - | + | ||
| - | </font> | + | |
<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | 14.52μL ddH2O</span></font><br> | ||
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | 2.0μL 10x PCR Buffer</span></font><br> | ||
<font color="#000000"> | <font color="#000000"> | ||
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 1.6μL dNTPs</span></font><br> | |
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | 0.4μL Forward Primer (Prefix)</span></font><br> | ||
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | 0.4μL Reverse Primer (Suffix)</span></font><br> | ||
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | 1.0μL Template DNA</span></font><br> | ||
| + | <font color="#000000"><u> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | 0.08μL RTaq DNA Polymerase</span></u></font><br> | ||
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | 20.0μL Total</span></font></p> | ||
<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
| - | + | <font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"><u>Notes</u>: </span></font> | |
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<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
| + | <font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | ||
| + | </span></span></font><font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">The | ||
| + | mixture was pipetted into PCR tubes </span></font><br> | ||
| + | <font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | ||
| + | </span></span></font><font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">All | ||
| + | materials were kept on ice</span></font><br> | ||
<font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | <font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | ||
| - | + | </span></span></font><font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Colonies will inoculated into 5uL broth prior to PCR</span></font><br> | |
| + | <font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | ||
| + | </span></span></font><font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | Prefix and Suffix were used as Forward and Reverse Primers respectively | ||
| + | while amplifying standard biobrick parts</span></font><br> | ||
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma">- </span> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">PCR | ||
| + | Reaction: 95°C - 10 min</span></font></p> | ||
<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | | |
| - | + | 95°C - 30 sec</span></font><br> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
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| | ||
57°C - 30 sec (appropriate annealing temperature for prefix and | 57°C - 30 sec (appropriate annealing temperature for prefix and | ||
| - | suffix)</span></font>< | + | suffix)</span></font><br> |
| - | + | ||
<font color="#000000"> | <font color="#000000"> | ||
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | | |
| - | + | 72°C - 30 sec </span> | |
| - | + | </font><br> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | | |
| - | + | (<i>28 cycles all together)</i></span></font><br> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | | |
| - | + | 72°C - 5 min </span> | |
| - | + | </font></p> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left"><font color="#000000"><u> | |
| - | + | <a name="Agarose_Gel_Electrophoresis_"><span style="font-size: 12pt; font-family: Trebuchet MS; font-style: italic">Agarose Gel Electrophoresis</span></a></u></font><a name="Agarose_Gel_Electrophoresis_"> | |
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | + | </a> |
| - | + | ||
| - | + | ||
| - | <span style="font-size: 12pt; font-family: Trebuchet MS; font-style: italic"> | + | |
| - | + | ||
<ul> | <ul> | ||
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Prepare a 2% or 1% Agarose Gel (amount in grams depending on volume | |
| - | + | of TAE buffer used). Add 0.1% Ethidium Bromide of the total volume. | |
| - | + | </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Place the gel in the Electrophoresis Apparatus with the wells facing | |
| - | + | the Negative Electrode. </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Fill the apparatus with 1% TAE Buffer to fully submerge the wells. | |
| - | + | </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Load 5µL of 1kb Ladder for each Run </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Add 0.1% of 10% Loading Dye to the respective volume of sample. Mix | |
| - | + | well and spin down. </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Pipette the samples into the wells and run at 106 Volts. </span> | |
| - | + | </font></li> | |
| - | + | </ul> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left; "> | |
| - | + | <font color="#000000"><u><font size="3"> | |
| - | + | <span style="font-family: Trebuchet MS; font-style: italic"> | |
| - | + | <a name="Gel_Purification_of_DNA_(Qiagen_QIAquick_Gel_Extraction_Kit)">Gel | |
| - | + | Purification of DNA (Qiagen QIAquick Gel Extraction Kit)</a></span></font></u></font></p> | |
| - | + | ||
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left; | + | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
<ul> | <ul> | ||
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Exercise the DNA fragment from the Agarose Gel using a scalpel. | |
| - | + | Minimize the extra peripheral gel slice. </span></font></li> | |
| - | + | <li> | |
| - | + | <font face="Tahoma" size="2" color="#000000">Weigh the </font> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | gel slice (0.1g = 100uL) and add 3 Volumes of Buffer QG to every 1 | |
| - | + | Volume of Gel. </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Incubate in 50°C water bath for 10 minutes to completely dissolve | |
| - | + | the gel slice.</span></font></li> | |
| - | + | <li> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400">Add 1 Volume of | |
| - | + | Isopropanol to the sample. Mix well</span></font></li> | |
| - | + | <li> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400">Apply the sample | |
| - | + | to the QIAquick column. Centrifuge at 13,500 rpm for 1 minute. | |
| - | + | [Repeat till the total volume of the sample has sieved through the | |
| - | + | column]. Discard the flow through</span></font></li> | |
| - | + | <li> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400">Apply </span> | |
| - | + | </font><font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 0.5mL Buffer QG to the QIAquick column. Centrifuge at 13,500 rpm for | |
| - | + | 1 minute.</span></font></li> | |
| - | + | <li> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400">Discard the flow | |
| - | + | through</span></font></li> | |
| - | + | <li> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400">Wash the column | |
| - | + | with 0.75mL Buffer PE. Centrifuge at 13,500 rpm for 1 minute.</span></font></li> | |
| - | + | <li> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400">Discard the flow | |
| - | + | through and Centrifuge at 13,500 rpm for an additional 1 minute to | |
| - | + | eliminate any residual ethanol.</span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Place the QIAquick column into a 1.5mL Eppendorf tube.</span></font></li> | |
| - | + | <li> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | <span style="font-family: Tahoma; font-weight: 400">Apply the </span> | |
| - | + | </font><font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 30uL warm H<sub>2</sub>O to the column. Let the column stand for at | |
| - | + | least 1 minute.</span></font></li> | |
| - | + | <li> | |
| - | + | <font size="2" color="#000000"> | |
| - | + | <span style="font-weight: 400; font-family: Tahoma">Centrifuge at | |
| - | + | 13,500 rpm for 1 minute.<br> | |
| - | + | </span></font></li> | |
| - | + | </ul> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left; margin-left: 0pt; margin-right: 10.0pt; "> | |
| - | + | <font color="#000000"><u> | |
| - | + | <span style="font-size: 12pt; font-family: Trebuchet MS; font-style: italic"> | |
| - | + | <a name="Determining_DNA_Concentration_Using_NanoDrop_Spectrophotometry">Determining DNA Concentration Using NanoDrop Spectrophotometry</a></span></u></font></p> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | </span></font></li> | + | |
| - | + | ||
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left; margin-left: 0pt; margin-right: 10.0pt; | + | |
| - | + | ||
| - | + | ||
| - | + | ||
<ul> | <ul> | ||
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Choose the Nucleic Acid Measurement option in the programme. </span> | |
| - | + | </font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Initialize the NanoDrop by adding 1uL clean H<sub>2</sub>O. Clean | |
| - | + | the sensor gently with tissue. </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Set Blank by adding an additional 1uL of clean H<sub>2</sub>O. Wipe | |
| - | + | off. </span></font></li> | |
| - | + | <li> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Add 1uL of the DNA sample to be measured. Wipe off after each run. | |
| - | + | </span></font> </li> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
</ul> | </ul> | ||
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left; margin-left: 0pt; margin-right: 10.0pt; "> | |
| - | + | <font color="#000000"><u> | |
| - | + | <span style="font-size: 12pt; font-family: Trebuchet MS; font-style: italic"> | |
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left; margin-left: 0pt; margin-right: 10.0pt; | + | <a name="DNA_Digestion_">DNA Digestion </a> </span></u></font></p> |
| - | + | ||
| - | + | ||
| - | + | ||
<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
<font color="#000000"> | <font color="#000000"> | ||
<span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| - | __μL ddH2O (to a total of 40uL)</span></font>< | + | __μL ddH2O (to a total of 40uL)</span></font><br> |
| - | + | ||
<font color="#000000"> | <font color="#000000"> | ||
<span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| - | 4μL 10X NEBuffer</span></font>< | + | 4μL 10X NEBuffer</span></font><br> |
| - | + | ||
<font color="#000000"> | <font color="#000000"> | ||
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 0.4μL 100X BSA</span></font><br> | |
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">1μg | ||
| + | DNA Sample </span></font><br> | ||
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | __μL 1<sup>st</sup> Restriction Enzyme</span></font><br> | ||
| + | <font color="#000000"><u> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | __μL 2<sup>nd</sup> Restriction Enzyme (optional)</span></u></font><br> | ||
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | 40μL Total</span></font></p> | ||
| + | <p class="MsoNormal" style="text-autospace: none; text-align: left"><font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400; text-decoration: underline">Notes: | ||
| + | </span></font></p> | ||
| + | <p class="MsoNormal" style="text-autospace: none; text-align: left"><font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">- | ||
| + | The NEB official website should be checked for buffers suitable for each | ||
| + | restriction enzyme. Results can vary depending on double or single | ||
| + | digestion. </span></font><br> | ||
| + | <font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | ||
| + | </span></span></font><font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | Incubate the digestion sample at 37°C for 3 hours (digestion time can | ||
| + | also vary depending on enzyme). Prolonged digestion may lead to Star | ||
| + | Activity otherwise. </span></font><br> | ||
| + | <font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | ||
| + | </span></span></font><font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">The | ||
| + | volume of DNA must be calculated from its concentration. In restriction | ||
| + | digestion test, the minimum volume that is equals 1ug DNA can be | ||
| + | utilized. However, for purification, a much greater volume of DNA should | ||
| + | be used. </span></font><br> | ||
| + | <font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | ||
| + | </span></span></font><font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | Appropriate amount of enzyme is derived from its concentration and the | ||
| + | fact that 5 units of enzyme digest 1ug DNA.</span></font></p> | ||
<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
| - | + | <font color="#000000"> | |
| - | + | <a name="Dephosphorylation_of_5_Ends_of_Vector_Backbone"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> </span><span style="font-size: 12pt; font-family: Trebuchet MS; font-style: italic"><u>Dephosphorylation of 5' Ends of Vector Backbone</u></span></a></font></p> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left"><font face="Tahoma" size="2" color="#000000">- </font> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">Add | |
| - | + | 0.5µL (0.5 units per 1 ug DNA) of Calf Intestinal Alkaline Phosphatase | |
| - | + | (CIAP) to the digested sample</span></font><font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | immediately after digestion.<br> | |
| - | + | - In</span></font><font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">cubate at 37°C for 30 minutes</span></font><font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> </span></font></p> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left; margin-left: 0pt; margin-right: 10.0pt; "> | |
| - | + | <font color="#000000"><u> | |
| - | + | <span style="font-size: 12pt; font-family: Trebuchet MS; font-style: italic"> | |
| - | + | <a name="Vector-Insert_Ligation">Vector-Insert Ligation</a></span></u></font></p> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | <p class="MsoNormal" style=" | + | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left; margin-left: 0pt; margin-right: 10.0pt; | + | |
| - | + | ||
| - | + | ||
| - | + | ||
<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
<font color="#000000"> | <font color="#000000"> | ||
<span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">__μL | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">__μL | ||
| - | Autoclaved ddH2O (to a total of 20uL)</span></font>< | + | Autoclaved ddH2O (to a total of 20uL)</span></font><br> |
| - | + | ||
<font color="#000000"> | <font color="#000000"> | ||
<span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| - | 2μL T4 Ligase Buffer</span></font>< | + | 2μL T4 Ligase Buffer</span></font><br> |
| - | + | ||
<font color="#000000"> | <font color="#000000"> | ||
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">1μL | |
| - | + | T4 DNA Ligase</span></font><br> | |
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | __μL Vector DNA</span></font><br> | ||
| + | <font color="#000000"><u> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">__μL | ||
| + | Insert DNA</span></u></font><br> | ||
| + | <font color="#000000"> | ||
| + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| + | 20μL Total</span></font></p> | ||
<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
| - | + | <font color="#000000"><u> | |
| - | + | <span style="text-decoration: none; font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | </span></u></font><font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400; text-decoration: underline">Notes:</span></font></p> | |
| - | + | <p class="MsoNormal" style="text-autospace: none; text-align: left; "> | |
| - | + | <font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | |
| - | + | </span></span></font><font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Insert and Vector must be in a 3:1 ratio. The amount of each depends on | |
| - | + | their concentration (ng/uL) and legnth (bp). </span></font><br> | |
| - | + | <font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left; | + | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
I</span></span></font><font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">ncubate | I</span></span></font><font color="#000000"><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">ncubate | ||
at room temperature for 1 hour. </span></font></p> | at room temperature for 1 hour. </span></font></p> | ||
| - | <p class="MsoNormal" style="text-autospace: none; text-align: left; | + | <p class="MsoNormal" style="text-autospace: none; text-align: left; "><font color="#000000"> |
| - | + | <span style="font-size: 12pt; font-family: Trebuchet MS; font-style: italic; text-decoration: underline"> | |
| - | + | <a name="PCR_Deletion_(Site-Directed_Mutagenesis)_Reaction">PCR Deletion (Site-Directed Mutagenesis) Reaction</a></span></font></p> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
| - | + | <font color="#000000"><u> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Note</span></u><span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">: | |
| - | + | Keep everything on ice and add all volumes in a PCR tube.</span></font></p> | |
<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
<font color="#000000"> | <font color="#000000"> | ||
<span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">? | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">? | ||
µL ddH2O (? = whatever volume needed to bring the total volume up to | µL ddH2O (? = whatever volume needed to bring the total volume up to | ||
| - | 50µL)</span></font>< | + | 50µL)</span></font><br> |
| - | + | ||
<font color="#000000"> | <font color="#000000"> | ||
<span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| - | 5.0μL 10x PfuUltra buffer</span></font>< | + | 5.0μL 10x PfuUltra buffer</span></font><br> |
| - | + | ||
<font color="#000000"> | <font color="#000000"> | ||
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 1.0μL dNTPs</span></font><br> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">? | |
| - | + | uL forward primer = 125ng fwd primer ÷ fwd primer concentration (ng/µL)</span></font><br> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">? | |
| - | + | uL reverse primer = 125ng rvs primer ÷ rvs primer concentration (ng/µL)</span></font><br> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400">? | |
| - | + | µL dsDNA= 20ng insert ÷ insert concentration (ng/µL)</span></font><br> | |
| - | + | <font color="#000000"><u> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 1.0μL </span></u> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | PfuUltra<u> high-fidelity DNA polymerase</u></span></font><br> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 50.0μL Total<br> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
</span></font></p> | </span></font></p> | ||
| - | <p class="MsoNormal" style="text-indent: -36.0pt; text-autospace: none; text-align: left; margin-left: 36.0pt; margin-right: 0pt; | + | <p class="MsoNormal" style="text-indent: -36.0pt; text-autospace: none; text-align: left; margin-left: 36.0pt; margin-right: 0pt; "> |
| - | + | <font size="2" color="#000000"><span style="font-family: Tahoma">-<span style="font-weight: 400"> | |
| - | + | </span></span></font><font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | Volumes of diluted primer based on calculations for our ng/µL | |
| - | + | concentrations</span></font></p> | |
<p class="MsoNormal" style="text-autospace: none; text-align: left"> | <p class="MsoNormal" style="text-autospace: none; text-align: left"> | ||
<font color="#000000"> | <font color="#000000"> | ||
<span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| - | 95°C for 2min</span></font>< | + | 95°C for 2min</span></font><br> |
| - | + | ||
<font color="#000000"> | <font color="#000000"> | ||
<span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | ||
| - | 95°C for 30sec (18 times)</span></font>< | + | 95°C for 30sec (18 times)</span></font><br> |
| - | + | ||
<font color="#000000"> | <font color="#000000"> | ||
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 55°C for 30sec</span></font><br> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 72°C for 1 min/kb</span></font><br> | |
| - | + | <font color="#000000"> | |
| - | + | <span style="font-size: 10pt; font-family: Tahoma; font-weight: 400"> | |
| - | + | 1min/kb corresponds to: 3.20min (RFP), 3.50min (VioA), 5.40min (VioB), | |
| - | + | 3.00min (VioE)</span></font></p></td> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
</tr> | </tr> | ||
| - | + | </table> | |
| - | + | </div> | |
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Latest revision as of 01:02, 27 September 2012
"
Team:HKU HK
From 2011.igem.org
Molecular Cloning Protocols
|
Transforming Competent E.coli with the
Plasmid:
Notes:
-
Colonies grown on plate were selected for colony PCR screening. Miniprep to Extract the Plasmid from E.coli (QIAprep Spin Miniprep Kit):
Midiprep for Large Volumes of Culture (QIAGEN Plasmid Midi Kit): Refer to the QIAGEN website for the protocol. PCR Amplification of Gene from Bacterial Genome/Standard Biobrick Parts
37.5μL ddH2O - For sequential PCR reactions: After the first PCR is complete, perform PCR clean-up. Then use the product to conduct the second PCR reaction, after which gel purification needs to be carried out. - The volume of DNA used in the second PCR reaction depends on the concentration of the DNA after the PCR Clean-up. PCR Clean-Up – Qiagen QIAquick PCR Purification:
14.52μL ddH2O Notes:
-
The
mixture was pipetted into PCR tubes
95°C - 30 sec
Gel Purification of DNA (Qiagen QIAquick Gel Extraction Kit)
Determining DNA Concentration Using NanoDrop Spectrophotometry
__μL ddH2O (to a total of 40uL) Notes:
-
The NEB official website should be checked for buffers suitable for each
restriction enzyme. Results can vary depending on double or single
digestion. Dephosphorylation of 5' Ends of Vector Backbone -
Add
0.5µL (0.5 units per 1 ug DNA) of Calf Intestinal Alkaline Phosphatase
(CIAP) to the digested sample
immediately after digestion.
__μL
Autoclaved ddH2O (to a total of 20uL) Notes:
-
Insert and Vector must be in a 3:1 ratio. The amount of each depends on
their concentration (ng/uL) and legnth (bp). PCR Deletion (Site-Directed Mutagenesis) Reaction Note: Keep everything on ice and add all volumes in a PCR tube.
?
µL ddH2O (? = whatever volume needed to bring the total volume up to
50µL) - Volumes of diluted primer based on calculations for our ng/µL concentrations
95°C for 2min |
Our kind Sponsors:
