- Purchase BACE2 plasmid (http://dnasu.org/DNASU/GetCloneDetail.do?cloneid=76140)
- Design primers for BACE2 and EYFP
- Run PCR on BACE2 and EYFP
- Perform golden gate on PCR products and GGDonr
- Perform LR reaction with products from previous step, TRE-t, and backbone
- Transfect with expression vector from previous step and rtta vector
Observe for fluorescence in presence of Dox (more work needed on details of actual experiment)
We hope to observe yellow fluorescence on the membrane of the cell in the presence of Dox
Case 1:
YFP visible on surfaceCase 2:
YFP visible
| Possible Results | Possible Explanation | Controls | Protocols | Parts |
|---|---|---|---|---|
YFP visible on surface | success | Image cell with out Dox. No fluorescence should be present | PCR | Primers (AL1-4), BACE2 plasmid, eYFP plasmid |
| YFP visible in the cell | YFP not bound to BACE, YFP disrupts BACE signal sequence | GoldenGate | PCR products, GGDonr | |
| YFP visible out of cell | YFP bound to incorrect terminal of BACE | LR | GG product, TRE-t, backbone | |
| No YFP visible | no Dox, cloning mistake | Transfect | LR product, rTTA vector | |
| Image |