Diagram:
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- 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 as well as HEF1a + mkate
- 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 | Done? |
|---|---|---|---|---|---|
YFP visible on surface | success |
| 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 (Gateway) (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 | ||
| no mkate visible | transfection error | Assay Bace2 activity | |||
| Image |
Options for Directly Assaying BACE2 Expression
| Link | Cost | Comments |
|---|---|---|
| https://www.anaspec.com/products/product.asp?id=57836 | $435 | This is a kit to directly measure BACE2 expression. Call tech support. |
| http://www.researchgate.net/post/Which_are_the_dye_suitable_for_visualization_of_neuron_with_beta-amyloid_protein_and_plaque10 | ||
| atomic force microscopy, Pittsburgh compound B |
Data
6/17:
Nanodrop from YFP PCR products with original primers :
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299 ng/uL |