Steps
The phase 1 project can be broken down into these steps:
Order DNA from IDT (approx 10.6kb)
Phosphorylate and ligate blunt ends (use NEB SDM kit)
Transformation DH5 alpha cells, plate on LB + 100 ug/mL ampicillin
Pick AmpR colonies and miniprep, screen colonies using a restriction enzyme that cuts 2 - 5 times to create diagnostic fragments
Choose 2-3 clones for sequencing using Oxford Nanopore Technology (ONT)
Maxiprep using kit to produce ultrapure plasmid
Begin culturing HEKa (Primary Epidermal Keratinocytes)
Transform HEKa with plasmids using Lipofectamine DNA Transfection Reagent
Determine transfection efficiency with fluorescence microscopy for presence of EGFP 1 - 4 days after transfection
a) Control plasmid/HEKa
b) GC plasmid/HEKa
10. Optimize lipofection efficiency
11. (Optional) Prepare complementary DNA (cDNA) and perform Reverse Transcriptase PCR (RT-PCR)
a) Run qPCR on MDM2, EGFP and reference gene
12. Prepare whole cell protein lysates
a) run SDS-PAGE transfer PAGE to nitrocellulose filter,
b) add horseradish peroxidase (HRP) conjugated mouse anti-human MDM2, EGFP and reference protein TBD
13. Start cancer cell line A431 culture (and possibly another cancer line TBD)
14. Transform HEKa and A431 (and other cancer line) with plasmids using Lipofectamine DNA Transfection Reagent
a) Control plasmid/HEKa
b) GC plasmid/HEKa
c) Control plasmid/A431
d) GC plasmid/A431
e) Control plasmid/another cancer line TBD
f) GC plasmid/another cancer line TBD
15. Determine transfection efficiency with fluorescence microscopy for presence of EGFP 1 - 4 days after transfection
16. (Optional) Prepare cDNA and perform Reverse Transcriptase PCR (RT-PCR)
a) Run qPCR on MDM2, EGFP and reference gene
17. Prepare whole cell protein lysates
a) run SDS-PAGE transfer PAGE to nitrocellulose filter,
b) add horseradish peroxidase (HRP) conjugated mouse anti-human MDM2, EGFP and reference protein TBD
18. TUNEL assay
19. Run Gel on healthy and cancer cell lines to look for evidence of apoptosis
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