June 9
Plan of Action
- Build hef1+lilrb2
- design cloning strategy
- acquire promoter
- acquire pdest
- build pentr lilrb2
- resuspend gblocks
- acquire gg domd
- run a golden gate
Questions:
- How do we turn a gblock into an entry vector?
- Can we get beta-amyloid in both monomer and oligomer form instead of just monomers that we would have to oligomerize?
- Do we need the cells to be expressing GFP?
June 10
Answers:
- We will work on that later today
- No preference
- Makes for pretty pictures and not hard to do since we're already transfecting a plasmid
June 11
| Making LilrB2/PirB expression vectors | Start Date | Progress/condition/next steps | Completed? (Date) | Result | Location of part (if needed, I don't know) |
|---|---|---|---|---|---|
Making pENTR from gBlocks: We have: We need: Golden-gating will result in: | 6/12 | GG mixtures in thermocycler GGDonr cell (culture?) in incubator Will make GGDonr Bacterial stock today (6/13) | |||
After that, Gibson (LR): Same for PirB We will get: 12_Hef1a_LilrB2 |
6/12/14
GoldenGate table:
| Add | Conc | Volm(x) | LilrB2 | PirB | Control | Order of adding | |
| PirB-N | 10ng/ul | 5ul | 0ul | 5ul | 0ul | 2 | |
| PirB-C | 10ng/ul | 5ul | 0ul | 5ul | 0ul | 2 | |
| LilrB2 | 10ng/ul | 5ul | 5ul | 0ul | 0ul | 2 | |
| GGDonr | 44.8ng/ul | 2.23ul | 2.23ul | 2.23ul | 2.23ul | 2 | |
| 10x T4 Ligase Buffer | 2ul | 2ul | 2ul | 2ul | 3 | ||
| BSA | 2ul | 2ul | 2ul | 2ul | 3 | ||
| BsaI (enzyme) | 1ul | 1ul | 1ul | 1ul | 4 | ||
| T4 Ligase (enzyme) | 1ul | 1ul | 1ul | 1ul | 3 | ||
| H20 | 6.77ul | 1.77ul | 11.77ul | 1 | |||
| Total | 20ul | 20ul | 20ul | 20ul |
| 1 | LilrB2 |
| 2 | PirB |
| 3 | Control |