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Team:HKU HongKong/Data/Weekly Notebook.html
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| - | + | <p style="text-align: justify"> </p> | |
| + | <h2 style="font-variant: normal; vertical-align: baseline; clear: left; color: #232323; font-family: Gentium Basic; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: justify; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; margin: 0.7em 0px; padding: 0px"> | ||
| + | <span style="font-weight: 400; text-decoration: underline"> | ||
| + | <font face="Trebuchet MS" size="6"> Daily Lab Notebookd</font></span></h2> | ||
| + | <font face="Trebuchet MS" size="4"><br> | ||
| + | <a href="https://www.dropbox.com/s/x8jcmbsypox3ekv/Daily Lab Notebook.docx?m" target="new">Download</a><br>Weeks 1&2:</font></span></h2> | ||
| + | <p style="text-align: justify; font-style: normal; font-variant: normal; font-weight: normal; font-size: 13px; vertical-align: baseline; color: rgb(85, 85, 85); font-family: Lato, Tahoma, Arial, sans-serif; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; margin-left: 0px; margin-right: 0px; margin-top: 0px; margin-bottom: 20px; padding: 0px"> | ||
| + | </p> | ||
| + | <h2 style="margin:0.7em 0px; text-align: justify; font-variant: normal; vertical-align: baseline; color: #232323; font-family: Gentium Basic; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; padding: 0px; clear:left"> | ||
| + | <span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; display: inline !important; float: none; background-color: rgb(255, 255, 255)"> | ||
| + | 1)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b> Amplification of pvdQ gene using PCR from Pseudomonas aeuriginosa genomic DNA. The designed primers contain the prefix and suffix so that typical digestion for insertion into iGEM’s standard vector can be subsequently performed.</b></font><br> | ||
| + | <br> | ||
| + | <br> | ||
| + | </span><span style="font-size: 10pt; font-family: Tahoma">2)</span><font color="#232323" size="2" face="Tahoma"><b>Test digestion of amplified pvdQ with EcoRI only, EcoRI & SpeI, PstI, and EcoRI & XbaI. </b></font></h2> | ||
| + | <p style="text-align: justify"><span style="font-size: 10pt; font-family: Tahoma">3)</span><font color="#232323" size="2" face="Tahoma"><b> Repeat the digestion of pvdQ with EcoRI and SpeI multiple times to obtain the desirable conditions of the digestion to prevent Star Activity. </b></font> | ||
| + | </h2></p> | ||
| + | <br> | ||
| + | <p style="text-align: justify"><span style="font-size: 10pt; font-family: Tahoma">4)</span><font color="#232323" size="2" face="Tahoma"><b>Mass single digestion of the amplified pvdQ with EcoRI. Desirable fragment size should be about 5,600bp. The fragments that result due to Star activity are 3,800 and 1,800bp.</b></font> | ||
| + | </h2> | ||
| + | |||
| + | </p><br> | ||
| + | <p style="text-align: justify"><span style="font-size: 10pt; font-family: Tahoma">5)</span><font color="#232323" size="2" face="Tahoma"><b>Gel purification and Nano-drop concentration check of digested pvdQ.</b></font> | ||
| + | </h2> | ||
| + | </p> | ||
| + | <p style="text-align: justify"><font size="2" face="Tahoma" color="#232323"><br> | ||
| + | </font></p> | ||
| + | |||
| + | <div align="left"><font face="Trebuchet MS" size="4">Weeks 3:</font></span></h2> | ||
| + | <br> | ||
| + | <font face-"trebuchet MS" size="3">28/07/2012<font></span></h2> | ||
| + | <br> | ||
| + | 1)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Transform J23119 (constitutive promoter) and B0034 (Ribosomal Binding Site) from the kit. </b></font><br> | ||
| + | <br> | ||
| + | 2)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Culture colonies from the transformation of ligated pvdQ-B0015.</b></font><br> | ||
| + | |||
| + | </div> | ||
| + | <div align="left"><br> | ||
| + | <font face-"trebuchet MS" size="3">28/07/2012<font></span></h2> | ||
| + | <br> | ||
| + | 1)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Culture colonies from J23119 and B0034 transformation.</b></font><br> | ||
| + | <br> | ||
| + | 2)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Colony PCR to confirm successful ligation of pvdQ-B0015.</b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 3)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Test digestion with enzymes</b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 4)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Gel electrophoresis of the digested volume of DNA (about 1ug) <br> | ||
| + | to check correct band size.</b></font><br> | ||
| + | <br> | ||
| + | |||
| + | <font face-"trebuchet MS" size="3"> 30/07/2012<font></span></h2> | ||
| + | <br> | ||
| + | 1)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Miniprep the confirmed culture to extract J23119 and B0034 plasmid. </b></font><br> | ||
| + | <br> | ||
| + | 2)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Restriction digestion with EcoRI and SpeI of J23119. Desired fragment size is 32 bp. </b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 3)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.</b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 4)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Gel electrophoresis to cut out correct fragment size. <br> | ||
| + | Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment. | ||
| + | </b></font><br> | ||
| + | <br> | ||
| + | <font face-"trebuchet MS" size="3">31/07/2012<font></span></h2> | ||
| + | <br> | ||
| + | 1)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Redo digestion of the J23119 plasmid using a greater amount of starting DNA (20uL) in a 50uL digestion reaction. </b></font><br> | ||
| + | <br> | ||
| + | 2)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Perform electrophoresis and correct out the now-visible fragment of correct size and acceptable intensity.</b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 3)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Gel purify the J23119 fragment.</b></font><br> | ||
| + | |||
| + | <br> | ||
| + | |||
| + | <font face-"trebuchet MS" size="3"> 02/08/2012<font></span></h2> | ||
| + | <br> | ||
| + | 1)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Use nanodrop to check the concentration of previously digested B0034 as well as J23119. </b></font><br> | ||
| + | <br> | ||
| + | 2)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Set up a suitable digestion reaction with J23119 as the insert and B0034 as the vector. Perform the ligation and incubate at room temperature for one hour. </b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 3)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Transform the ligated product.</b></font><br> | ||
| + | |||
| + | <br> | ||
| + | <font face-"trebuchet MS" size="3"> 03/08/2012<font></span></h2> | ||
| + | <br> | ||
| + | 1)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Culture the visible colonies on the transformed plate. | ||
| + | Result: The transformed plate had minisatellite colonies. Hence very few colonies could be picked.<br> | ||
| + | Result: The transformed plate had minisatellite colonies. Hence very few colonies could be picked. | ||
| + | </b></font><br> | ||
| + | <br> | ||
| + | 2)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Perform colony PCR. <br> | ||
| + | Result: Expected amplified product’s size is similar to the band produced from primer dimerization. Hence the presence of the amplified product cannot be confirmed. | ||
| + | </b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 3)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.</b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 4)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Gel electrophoresis to cut out correct fragment size. <br> | ||
| + | Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment. | ||
| + | </b></font><br> | ||
| + | |||
| + | <font face-"trebuchet MS" size="3"> 30/07/2012<font></span></h2> | ||
| + | <br> | ||
| + | 1)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Miniprep the confirmed culture to extract J23119 and B0034 plasmid. </b></font><br> | ||
| + | <br> | ||
| + | 2)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Restriction digestion with EcoRI and SpeI of J23119. Desired fragment size is 32 bp. </b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 3)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.</b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 4)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Gel electrophoresis to cut out correct fragment size. <br> | ||
| + | Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment. | ||
| + | </b></font><br> | ||
| + | |||
| + | <font face-"trebuchet MS" size="3"> 30/07/2012<font></span></h2> | ||
| + | <br> | ||
| + | 1)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Miniprep the confirmed culture to extract J23119 and B0034 plasmid. </b></font><br> | ||
| + | <br> | ||
| + | 2)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Restriction digestion with EcoRI and SpeI of J23119. Desired fragment size is 32 bp. </b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 3)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.</b></font><br> | ||
| + | |||
| + | <br> | ||
| + | 4)</span><span style="color: rgb(35, 35, 35); font-family: Lato, Tahoma, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; "> | ||
| + | </span><font face="Tahoma" size="2"><b>Gel electrophoresis to cut out correct fragment size. <br> | ||
| + | Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment. | ||
| + | </b></font><br> | ||
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Latest revision as of 03:31, 27 September 2012
Team:HKU HK
From 2011.igem.org
Daily Lab Notebookd
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Weeks 1&2:
1)
Amplification of pvdQ gene using PCR from Pseudomonas aeuriginosa genomic DNA. The designed primers contain the prefix and suffix so that typical digestion for insertion into iGEM’s standard vector can be subsequently performed.
2)Test digestion of amplified pvdQ with EcoRI only, EcoRI & SpeI, PstI, and EcoRI & XbaI.
3) Repeat the digestion of pvdQ with EcoRI and SpeI multiple times to obtain the desirable conditions of the digestion to prevent Star Activity.
4)Mass single digestion of the amplified pvdQ with EcoRI. Desirable fragment size should be about 5,600bp. The fragments that result due to Star activity are 3,800 and 1,800bp.
5)Gel purification and Nano-drop concentration check of digested pvdQ.
Weeks 3:
28/07/2012
1) Transform J23119 (constitutive promoter) and B0034 (Ribosomal Binding Site) from the kit.
2) Culture colonies from the transformation of ligated pvdQ-B0015.
28/07/2012
1) Transform J23119 (constitutive promoter) and B0034 (Ribosomal Binding Site) from the kit.
2) Culture colonies from the transformation of ligated pvdQ-B0015.
28/07/2012
1) Culture colonies from J23119 and B0034 transformation.
2) Colony PCR to confirm successful ligation of pvdQ-B0015.
3) Test digestion with enzymes
4) Gel electrophoresis of the digested volume of DNA (about 1ug)
to check correct band size.
30/07/2012
1) Miniprep the confirmed culture to extract J23119 and B0034 plasmid.
2) Restriction digestion with EcoRI and SpeI of J23119. Desired fragment size is 32 bp.
3) Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.
4) Gel electrophoresis to cut out correct fragment size.
Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment.
31/07/2012
1) Redo digestion of the J23119 plasmid using a greater amount of starting DNA (20uL) in a 50uL digestion reaction.
2) Perform electrophoresis and correct out the now-visible fragment of correct size and acceptable intensity.
3) Gel purify the J23119 fragment.
02/08/2012
1) Use nanodrop to check the concentration of previously digested B0034 as well as J23119.
2) Set up a suitable digestion reaction with J23119 as the insert and B0034 as the vector. Perform the ligation and incubate at room temperature for one hour.
3) Transform the ligated product.
03/08/2012
1) Culture the visible colonies on the transformed plate. Result: The transformed plate had minisatellite colonies. Hence very few colonies could be picked.
Result: The transformed plate had minisatellite colonies. Hence very few colonies could be picked.
2) Perform colony PCR.
Result: Expected amplified product’s size is similar to the band produced from primer dimerization. Hence the presence of the amplified product cannot be confirmed.
3) Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.
4) Gel electrophoresis to cut out correct fragment size.
Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment.
30/07/2012
1) Miniprep the confirmed culture to extract J23119 and B0034 plasmid.
2) Restriction digestion with EcoRI and SpeI of J23119. Desired fragment size is 32 bp.
3) Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.
4) Gel electrophoresis to cut out correct fragment size.
Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment.
30/07/2012
1) Miniprep the confirmed culture to extract J23119 and B0034 plasmid.
2) Restriction digestion with EcoRI and SpeI of J23119. Desired fragment size is 32 bp.
3) Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.
4) Gel electrophoresis to cut out correct fragment size.
Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment.
