Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminA"
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Final DNA concentrations of the 4 tubes of miniprep, measured with Nanodrop: | Final DNA concentrations of the 4 tubes of miniprep, measured with Nanodrop: | ||
<ul> | <ul> | ||
− | <li>tube HO pl. 1 = 431.1 ng/uL</li> | + | <li>tube HO-Poly-KanMX4-HO pl. 1 = 431.1 ng/uL</li> |
− | <li>tube HO pl. 2 = 313.6 ng/uL</li> | + | <li>tube HO-Poly-KanMX4-HO pl. 2 = 313.6 ng/uL</li> |
− | <li>tube HO pl. 3 = 366.4 ng/uL</li> | + | <li>tube HO-Poly-KanMX4-HO pl. 3 = 366.4 ng/uL</li> |
− | <li>tube HO pl. 4 = 261.7 ng/uL</li> | + | <li>tube HO-Poly-KanMX4-HO pl. 4 = 261.7 ng/uL</li> |
</ul> | </ul> | ||
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<ul> | <ul> | ||
<li>the PCR product of HO-Poly-KanMX4-HO, a plasmid from Addgene </li> | <li>the PCR product of HO-Poly-KanMX4-HO, a plasmid from Addgene </li> | ||
− | <li>PCR product of the gblock 1.1</li> | + | <li>PCR product of the gblock vA-1.1</li> |
− | <li>PCR product of the gblock 1.2</li> | + | <li>PCR product of the gblock vA-1.2</li> |
− | <li>PCR product of the gblock 2</li> | + | <li>PCR product of the gblock vA-2</li> |
− | <li>PCR product of the gblock 3</li> | + | <li>PCR product of the gblock vA-3</li> |
− | <li>PCR product of the gblock 4</li> | + | <li>PCR product of the gblock vA-4</li> |
</ul> | </ul> | ||
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<ul> | <ul> | ||
<li>15 uL Gibson master mix</li> | <li>15 uL Gibson master mix</li> | ||
− | <li>1 uL HO plasmid at 100 ng/uL</li> | + | <li>1 uL HO-Poly-KanMX4-HO plasmid at 100 ng/uL</li> |
<li>1 uL “gBlock mix”</li> | <li>1 uL “gBlock mix”</li> | ||
<li>3 uL water</li></ul> | <li>3 uL water</li></ul> | ||
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<h2>Plan of the PCR</h2> | <h2>Plan of the PCR</h2> | ||
<ul> | <ul> | ||
− | <li>gBlock 1.1 with primers o15.141 and o15.144</li> | + | <li>gBlock vA-1.1 with primers o15.141 and o15.144</li> |
− | <li>gBlock 1.2 with primers o15.119 and o15.120</li> | + | <li>gBlock vA-1.2 with primers o15.119 and o15.120</li> |
− | <li>gBlock 2 with primers o15.127 and o15.128</li> | + | <li>gBlock vA-2 with primers o15.127 and o15.128</li> |
− | <li>gBlock 3 with primers o15.129 and o15.130</li> | + | <li>gBlock vA-3 with primers o15.129 and o15.130</li> |
− | <li>gBlock 4 with primers o15.131 and o15.132</li> | + | <li>gBlock vA-4 with primers o15.131 and o15.132</li> |
<li>HO-Poly-KanMX4-HO with primers o15.135 and o15.143</li> | <li>HO-Poly-KanMX4-HO with primers o15.135 and o15.143</li> | ||
</ul> | </ul> | ||
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<li>1uL of each sample was nanodroped and the concentrations are shown below</li> | <li>1uL of each sample was nanodroped and the concentrations are shown below</li> | ||
</ul> | </ul> | ||
− | <h2>Case of the gBlock 3</h2> | + | <h2>Case of the gBlock vA-3</h2> |
because it does not amplify well and is showing signs of unspecific binding we are performing a gel purification on this gBlock. | because it does not amplify well and is showing signs of unspecific binding we are performing a gel purification on this gBlock. | ||
<h1>PCR and gel purification</h1> | <h1>PCR and gel purification</h1> | ||
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<h2>plan of the PCR</h2> | <h2>plan of the PCR</h2> | ||
<ul> | <ul> | ||
− | <li>gBlock 1.1 with primers o15.141 and o15.142</li> | + | <li>gBlock vA-1.1 with primers o15.141 and o15.142</li> |
− | <li>gBlock 1.2 with primers o15.119 and o15.120</li> | + | <li>gBlock vA-1.2 with primers o15.119 and o15.120</li> |
− | <li>gBlock 2 with primers o15.127 and o15.128</li> | + | <li>gBlock vA-2 with primers o15.127 and o15.128</li> |
− | <li>gBlock 3 with primers o15.129 and o15.130</li> | + | <li>gBlock vA-3 with primers o15.129 and o15.130</li> |
− | <li>gBlock 4 with primers o15.131 and o15.132</li> | + | <li>gBlock vA-4 with primers o15.131 and o15.132</li> |
<li>HO-Poly-KanMX4-HO with primers o15.135 and o15.143</li> | <li>HO-Poly-KanMX4-HO with primers o15.135 and o15.143</li> | ||
<br> | <br> | ||
<br> | <br> | ||
</ul> | </ul> | ||
− | We also tried a <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols/Gibson_Assembly">Gibson Assembly</a> again with the amplified gBlocks 1.1, 1.2, 2, 4 and the HO vector ... which failed miserably. | + | We also tried a <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols/Gibson_Assembly">Gibson Assembly</a> again with the amplified gBlocks vA-1.1, 1.2, 2, 4 and the HO-Poly-KanMX4-HO vector ... which failed miserably. |
<div class="column-left" align="left"><img src="https://static.igem.org/mediawiki/2015/b/bf/IgemparisbettencourtGibsonfailed.jpg" width="350px"> | <div class="column-left" align="left"><img src="https://static.igem.org/mediawiki/2015/b/bf/IgemparisbettencourtGibsonfailed.jpg" width="350px"> | ||
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</br></br> | </br></br> | ||
<h1 class="date">August 26th</h1> | <h1 class="date">August 26th</h1> | ||
− | result of the PCR of the gBlocks (from left to right) 1.1, 1.2, 2, 3, 4, HO and the last column is the ladder 1kb | + | result of the PCR of the gBlocks (from left to right) vA-1.1, 1.2, 2, 3, 4, HO-Poly-KanMX4-HO and the last column is the ladder 1kb |
<div class="column-left" align="left"><img src="https://static.igem.org/mediawiki/2015/0/09/Pcrofthegblock111234hobright.jpg" width="350px"> | <div class="column-left" align="left"><img src="https://static.igem.org/mediawiki/2015/0/09/Pcrofthegblock111234hobright.jpg" width="350px"> | ||
</div> | </div> | ||
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<div class="column-left" align="left"><img src="https://static.igem.org/mediawiki/2015/0/02/Gelfor113hojb.jpg" width="350px"> | <div class="column-left" align="left"><img src="https://static.igem.org/mediawiki/2015/0/02/Gelfor113hojb.jpg" width="350px"> | ||
</div> | </div> | ||
− | <div class="column-left" align="right">The results are showing traces of unspecific binding for 1.1 and 3. The HO band is alsmost invisible, predicting a low DNA concentration.</div> | + | <div class="column-left" align="right">The results are showing traces of unspecific binding for 1.1 and 3. The HO-Poly-KanMX4-HO band is alsmost invisible, predicting a low DNA concentration.</div> |
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
<div class="column-left" align="right"> | <div class="column-left" align="right"> |
Revision as of 13:15, 27 August 2015