<p>To evaluate the success of our assembly, we performed multiple tests. First, we conducted a colony PCR to get a general notion if the plasmid was integrated. Next, we miniprep the liquid cultures and conduct PCR to amplify sections of the construct, using different combinations of primers. We later would send them for sequencing using primers that were positioned inside the original backbone of pSEVA.</p>
<p><b> GA 1 & 2 </b></p>
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<p>After it was concluded that the assembly had failed, we reasoned that our inserts might be too large, making it hard for the construct to form. As well as the possibility that the fragments' overhangs were being altered by the PCR.</p>
<p><b> GA 1 & 2 </b></p>
<p>After performing 10 Gibson Assembly with no success, we considered that the full construct was too large to form. We decided to pivot and create a smaller construct using the first two fragments. Additionally, we were advised to pick more single colonies from the transformed plates, as there was a possibility that one might have a successful assembly.</p>
