- Hetmann and I made glycerol stocks of our two R0010 + E0241 cultures from yesterday.
- 100 µL 50% glycerol, 100 µL liquid culture
- Hetmann and I made minipreps of our two cultures, following the Qiagen protocol.
- Hetmann and I digested (with Xba and Pst1) our two R0010 + E0241 cultures from yesterday.
- Mix the following in each of the two tubes:
- 8 µL DNA
- 0.5 µL Xba + 0.5 µL Pst1 + 1 µL
- 2 µL from a stock of 1 µL Xba + 1 µL Pst1 + 2 µL dH20
- 0.25 µL BSA + 0.75 µL dH20
- 1 µL from a stock of 1 µL BSA + 3 µL dH20
- 2.5 µL buffer 3
- 11.5 µL dH20 to bring the total volume to 25 µL
- Incubate at 37C for 1 hour (in reality, we incubated for about 4 hours since we broke for lunch and I went to a long meeting afterwards).
- Several of us (David, Tiffany, Matt, Valerie, Katie, Lewis, and I, IIRC) had a meeting with Shawn to discuss designs for our DNA nanostructure.
- Note: DNA helix width 2nm, helical turn every 3.4nm (10.5 bp). DNA gets floppy when longer than 50nm (~150bp).
- We settled on a simple honeycomb cylinder, since we have high confidence we can make it, and it's not obvious which designs will perform better than others. At present, we will not design a honeycomb cube or a flat cube.
- The biggest remaining challenge is to design the lid.
- I made e-gels of Hetmann's and my digests. They didn't turn out very well (lanes 8 and 9). Now that I look back at my digest protocol, I realize that I forgot to heat shock our digests for 15 min at 80C.
- Combine 10 µL of each digest with 10 µL of dH20
- Insert into e-gel wells
- Run e-gel for 30 min
- I spent the rest of the day designing nanostructures with David, Tiffany, Matt, Katie, and Lewis. We made two designs based on a honeycomb cylinder: a large cylinder with two single-ply lids and a smaller cylinder with two double-ply lids. Our figures are written on the whiteboard in the 5th floor breakroom; I'll write them down tomorrow if no one else recorded them.