Biomod/2011/Harvard/HarvarDNAnos:Lab Notebook

Team Member Lab Notebook Pages

 * Nick Perkons
 * Sherrie Wang
 * Evan Wu
 * Shwinn Ricci
 * Adam Marblestone

To Do List

 * Order new M13 scaffold
 * Put up new protocols like page gels, nanodropping origami; TEM prep ; etc.
 * Figure out optimal oxidation conditions for disulfide bond creation - William and Peng say to drop disulfide linkers because they are not compatible with gold
 * Look into photocleavable spacers and incorporate into designs - order these staples


 * Obtain TEM training


 * Extract nanoparticle chain from gel cut-outs
 * Make new nanoparticles with Steve
 * Conjugate nanoparticles with DNA
 * Figure out best way to extract nanoparticle chain from unbound nanoparticles
 * Finish AuNP-DNA conjugation protocol and run tests on AuNP-DNA-DNA hybridization


 * Finish minimal box test
 * Plan a cargo loading system for box
 * TEM Evan's box on Thur/Fri
 * AFM Evan's box on Thur/Fri
 * gel purify Evan's box on Thur/Fri
 * Refold the box
 * Successfully image the folded box
 * Order more staples for box
 * make digital schematic of box with cargo loading system


 * Order new sphere staples
 * TEM sphere again with better staining
 * coordinate on SphereCAD goals/requirements and make a mockup of a basic UI
 * Find and use a better method for purification of sphere
 * Screen different gel band positions for gel purification of sphere
 * Evaluate and increase yield of correctly folded spheres by varying annealing schedule and buffer conditions
 * get to the point where we can fold & purify closed or open spheres with near 100% reliability
 * a sub task for this week is gel extraction of sphere bands followed by more AFM


 * test 3 ways to make transitions between open and closed states: azobenzene, strand displacement, photocleavable spacer
 * test these both on the sphere and on "mini-box"
 * observe transitions by BOTH gel and AFM for the sphere
 * try to quantitate our ability to make transitions between closed and open
 * on minibox we need to try to go through multiple close/open cycles, too


 * load the sphere with cargo

2011-06-23
Yin Lab Meeting:

Notes:
 * thiolated nanoparticle strand needs to be distinct from the disulphide strand (to allow conjugation to gold without breaking the disulphide - gold itself can competitively reduce disulphides)
 * use something other than DTT to cleave disulphide bonds if you want to NOT also cleave the thiol bond to gold
 * box foldings with AuNP cargo should be 2-step not 1-step
 * linking of 5' S-S strand directly to gold particle works with high efficiency. we have one of these for the 15-mer, but it is short, so gel separation won't be good. still, we can try it.
 * look into conditions necessary for restriction enzyme activity and the two other options (i forgot the names) that were suggested that could cleave downstream of a recognition sequence given that peng thought we would need about ten bases of pre cutexcess (which is a lot more than I expected)
 * caged DNA instead of azobenzene: impractical this summer since we have the azobenzene strand already
 * use Type II restriction enzyme which is an "offset cutter" to cut close to origami while keeping cut site a reasonable distance away from the origami

 