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Description: Making HEK293 expressing LilrB2 and PirB on the cell membrane. Will test membrane localization by using anti-LilrB2 and anti-PirB antibodies in Experiment 2. Plan:- Order LilrB2 and PirB gBlocks
- Simulate Golden Gate on Geneious and make sure it works
- Golden Gate gBlocks into ggDONR
- Transform bacteria with Golden Gate product and plate
- Pick colonies and grow in liquid culture
- Miniprep cells
- LR into pDEST
Status:- First and second set of liquid cultures didn't grow, possibly because:
- Too much media
- Plates were old, antibiotic possibly not working
- First set of plates didn't have any blue colonies
- Second set of plates grew blue and white colonies
- PirB didn't grow any white colonies
- Colonies picked for LilrB2
- Liquid culture grew
- Miniprepped - Product to be verified
- Golden Gate repeated
- cells transformed and plated
- +ve control didn't grow
- LilrB2 grew blue and white colonies
- PirB grew white colonies
- Both liquid cultures grew
- Miniprepped - Product to be verified
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Experiment 2: Testing membrane localization of LilrB2 and PirBExperiment 3: Oligomerizing affinity of beta-amyloid from beta-amyloid monomers Experiment 4: Testing binding of monomers and oligomers to the cells from Experiment 1 and Experiment 2to receptors