Team:UCLA/Notebook/Protein Cages/1 July 2015

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Phillip's notes:

Introduction: Today the project overview will be discussed during our general meeting with Dr. Kosuri. Fasih will then help us take our glycerol stock from Dr. Yeates and plate them to select for making a starter culture tomorrow.

Meetng notes: site direct mutagenesis with whole plasmid. Need DpnI

Try Gibson assembly: Need Gibson and PCR enzymes, DpnI (cuts methylated GATC to remove old plasmid)

Conclusions: Upon retrieving the clones, it was realized that they were in the -20 degree fridge instead of the -80 degree fridge. The entirety of the tube was used to plate the cells on an agar plate with ampicillin, with the help of Tristan. He did not work over a flame. Conclusions: If colonies grow, we will make a starter culture tomorrow, and the large culture Friday. Hopefully something grows on our plates :(

Intro: researched protocols for glycerol stock replating. Lab-wide meeting with Sri. Watched Tristan plate our glycerol stock (-80 is in 7718, -20 is in 7736). Further reading of Yeates' methods. Looked at dynamic light scattering and native page for size analyses of cage with and without thrombin cleavage. I think we could get away with doing a normal sds-page to see if thrombin cleaves the cage. However, we will first need to verify proper cage structure with another method (dls?).

Glycerol Protocols: Addgene looks like a good place for protocols. Streaking pattern is good to know. https://www.addgene.org/plasmid-protocols/create-glycerol-stock/

Lab Meeting Pertinent Notes: Sri said normal site directed mutagenesis could work and is worth a shot (primer anneals both 20 bp regions flanking insert nucleotides). We would need DpnI.

Glycerol Plating: Use a flame (Tristan didn't). Incubate overnight to find single colony.

Yeates' methods:

2013 (more recent) paper with optimized cage

http://pubs.acs.org/doi/suppl/10.1021/ja402277f/suppl_file/ja402277f_si_001.pdf

2012 paper http://www.sciencemag.org/content/suppl/2012/05/30/336.6085.1129.DC1/Lai.SM.pdf

Future: Lookk up DpnI usage and other methods of adding insert besides overhang pcr that we initially considered.

Tyler Lee --Wtleeiv 18:48, 1 July 2015 (CDT)