To Do:
Friday:
- Nanodrop pENTR (was not useful)
- Transform pENTR into bacteria
- Incubate over night
- (5pm) Add Kan, incubate GGDonr liq culture (16hrs)
...
Saturday:
- Remove GGDonr liq culture from incubator (9am)
- Make cell stock, store at -80 degrees
- Put rest of cell culture in 4 degrees
- Remove pENTR cultures from incubator (10:30am, Shinjini)
- Pick cells from pENTR colonies, put in liquid culture, put in incubator at 12pm
- Pick cells from pENTR colonies, put in liquid culture, put in incubator at 5:30pm (16 hours)
...
Sunday:
- Take pENTR colonies from incubator. Make cell stock. Put in fridge and freezer respectively. (10 am). Did not work.
...
Monday:
- Miniprep GGDonr (is that a good idea?)
- Pick Cells again and incubate (at 6pm)
...
Tuesday:
- Miniprep pENTRs
- Make 5 femptomolar working solution
- LR (~ 16 hours min, 24 max)
- PCR (possibly)
...
June 9
Plan of Action
- Build hef1+lilrb2
...
- 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 | 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 fridge (in case transformations did not work) | ||
After that, Gateway (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 |