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 pENTR from gBlocks:
We have:
Q1_LilrB2_Qx
Q1_PirB-N_Q2, Q2_PirB-C_Qx
We need:
pGGDONR : has Q1 and Qx sites, L1 and L2 sites
Golden-gating will result in:
pENTR_L1_LilrB2_L2 (order of L's correct, I checked!)
pENTR_L1_PirB2_L2
After that, Gibson (LR):
pENTR_L1_LilrB2_L2
pENTR_L4_Hef1a_R1 (not sure if this is the one we want)
pZDonor 1-GTW-2 (this is the DEST Shinjini used in her Geneious)
Same for PirB
pENTR_L1_PirB_L2
pENTR_L4_Hef1a_R1
pZDonor 1-GTW-2
We will get:
12_Hef1a_LilrB2
12_Hef1a_PirB