Team:UCLA/Notebook/Protein Cages/6 July 2015
Phillip's notes:
Introduction: For today, colony PCR primers will be designed in order to use them to verify that the correct plasmid and gene is inside our clones. The protocol will be laid out to do the procedure when reagents our moved to the Boelter lab. Preparation will also be made for protein expression this week.
Colony PCR primers:
The file “pET-22b with PCquad Optimized” was created by using a RE digestion and ligation reaction on Benchling. Primers were designed to overlap part of the PCquad gene and the vector. The primers designed were the following. Note PCquad gene was inserted reading right to left.
Forward: 5’-GAAGGTGTCATAATCGTATCCAGTCG-3’
Reverse: 5’- GGTGATGTCGGCGATATAGGC-3’
Notes: For reverse primer, a self-dimer with delta G = -5.19 kcal/mol formed, while the max delta G = -41.97 kcal/mol. A heterodimer forms with -6.66 kcal/mol while the max delta G is -45.9 kcal/mol. All other parameters are ok.
The colony PCR primer forward seems to anneal to the “PCquad original” everywhere except 3 base pairs. The order was placed from IDT.
Conclusions: Most of today was used in preparation for protein expression of the cage. It may be nice to give a presentation on the techniques involved for characterizing the protein cage, as well as to become acquainted with the theory. If possible, tomorrow will be used to do colony PCR to verify that the Yeates strain contains the proper plasmid and gene.