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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: 

  1. How do we turn a gblock into an entry vector?
  2. Can we get beta-amyloid in both monomer and oligomer form instead of just monomers that we would have to oligomerize?
  3. Do we need the cells to be expressing GFP?

June 10 

Answers:

  1. We will work on that later today
  2. No preference
  3. Makes for pretty pictures and not hard to do since we're already transfecting a plasmid

June 11

Making LilrB2/PirB expression vectors

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


 

 

 

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