Difference between revisions of "Team:UCLA/Notebook/Protein Cages/14 July 2015"

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Intro: Pondered how to express cage in iGEM compatible vector. Designed and ordered appropriate primers.
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Vinson had a plasmid that contains: T7 promoter-RBS-iGEM suffix. After much thought, I designed primers to amplify our g block with the iGEM prefix, and tack on a his tag, stop codon, and the suffix in two rounds of PCR. The forward primer will be the iGEM prefix and the first 20 nucleotides of the cage sequence. The first reverse primer will be the last 20 nucleotides of the cage and the his tag. The second reverse primer will inclund the final nucleotide of the cage, his tag, stop codin and suffix. This two step addition was the only way to add the his tag and stop codon without going over the 60bp limit imposed by IDT for ordering oligos. Sri said to express the cage in this new iGEM compatible vector, as well as the pET-22b vector in parallel, just to see which is better. He recommended that we initially transform the ligated plasmids into DHSalpha competent cells (in his lab) to verify correct plasmids before finally transforming them into BL21-DE3 competent cells for protein expression.

Revision as of 04:00, 15 July 2015

iGEM UCLA




Intro: Pondered how to express cage in iGEM compatible vector. Designed and ordered appropriate primers.

Vinson had a plasmid that contains: T7 promoter-RBS-iGEM suffix. After much thought, I designed primers to amplify our g block with the iGEM prefix, and tack on a his tag, stop codon, and the suffix in two rounds of PCR. The forward primer will be the iGEM prefix and the first 20 nucleotides of the cage sequence. The first reverse primer will be the last 20 nucleotides of the cage and the his tag. The second reverse primer will inclund the final nucleotide of the cage, his tag, stop codin and suffix. This two step addition was the only way to add the his tag and stop codon without going over the 60bp limit imposed by IDT for ordering oligos. Sri said to express the cage in this new iGEM compatible vector, as well as the pET-22b vector in parallel, just to see which is better. He recommended that we initially transform the ligated plasmids into DHSalpha competent cells (in his lab) to verify correct plasmids before finally transforming them into BL21-DE3 competent cells for protein expression.