Difference between revisions of "Team:KU Leuven/Research/Results"
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<p><b>Method 2</b><br/> | <p><b>Method 2</b><br/> | ||
− | Because our method was aberrant compared to the IDT protocol, a vector with correct overhangs was PCR amplified. This vector was used in a one-reaction Gibson assembly. After transformation, liquid cultures were grown from which the plasmid DNA was isolated. Restriction mapping was performed (Figure 14) and showed presence of only pUC19. | + | Because our method was aberrant compared to the IDT protocol, a vector with correct overhangs was PCR amplified. This vector was used in a one-reaction Gibson assembly. After transformation, liquid cultures were grown from which the plasmid DNA was isolated. Restriction mapping was performed (Figure 14) and showed presence of only pUC19. <br/> |
+ | |||
+ | <div class ="center"> | ||
+ | <div id="image13"> | ||
+ | <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/3/3a/KU_Leuven_RestrictionDigest.jpeg" | ||
+ | data-lightbox="example-set" data-title="Method1Test"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/3/3a/KU_Leuven_RestrictionDigest.jpeg" width='50%'></a> | ||
+ | <h4> | ||
+ | <div id=figure12>Figure 13</div></h4> | ||
+ | <div class="whiterow"></div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
To get rid of the template pUC, the PCR amplified backbone was digested with DpnI. This restriction enzyme only cuts methylated DNA and is thus inactive on PCR amplified pUC and synthesised gBlocks.<br/> | To get rid of the template pUC, the PCR amplified backbone was digested with DpnI. This restriction enzyme only cuts methylated DNA and is thus inactive on PCR amplified pUC and synthesised gBlocks.<br/> | ||
<br/> | <br/> | ||
Even after digestion with DpnI, pUC19 kept appearing (Figure ?).<br/> | Even after digestion with DpnI, pUC19 kept appearing (Figure ?).<br/> | ||
+ | <div class ="center"> | ||
+ | <div id="image14"> | ||
+ | <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/3/3d/KU_Leuven_GelSeparation.jpg" | ||
+ | data-lightbox="example-set" data-title="Method1Test"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/3/3d/KU_Leuven_GelSeparation.jpg" width='50%'></a> | ||
+ | <h4> | ||
+ | <div id=figure14>Figure 14</div></h4> | ||
+ | <div class="whiterow"></div> | ||
+ | </div> | ||
+ | </div> | ||
<br/> | <br/> | ||
To optimize the vector PCR, pUC19 was first linearized using the unique restriction enzyme XbaI. Again, the PCR product was digested with DpnI. This plasmid was also transformed as a negative control and did not show any positive colonies. The plates that should contain the insert did show positive colonies.</p> | To optimize the vector PCR, pUC19 was first linearized using the unique restriction enzyme XbaI. Again, the PCR product was digested with DpnI. This plasmid was also transformed as a negative control and did not show any positive colonies. The plates that should contain the insert did show positive colonies.</p> |
Revision as of 01:22, 19 September 2015
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