- 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
- Digest portion with Bal1, run gel and compare to virtual gel
Observe for fluorescence in presence of Dox (more work needed on details of actual experiment)
| 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 (digest and run on gel to confirm proper assembly) | PCR products, GGDonr | |
| YFP visible out of cell | YFP bound to incorrect terminal of BACE | LR (digest and run on gel to confirm proper assembly) | GG product, TRE-t, backbone | |
| No YFP visible | no Dox, cloning mistake | Transfect | LR product, rTTA vector | |
| Assay Bace2 activity | ||||
| Image |