<p>Lab 9</p><p>CPP Protein Delivery</p><p>1. Pipet 50 μg of R9 (350 μL of 100 μM solution) into 150 μL of Dulbecco’s phosphate buffer solution (PBS) 1X with no calcium or magnesium (pH 6.7) (500 μL total volume) into a 1.5 mL Eppendorf tube. Mix by repeatedly pipetting up and down. DO NOT VORTEX. 2. Similarly, in .5 mL tubes, pipet 50 μg of GUS Enzyme (50 μL of a 1:1 solution) into 450 μL of PBS. Repeat so that you have 500 μL of GUS in each. The dilutions will ensure the complex does not precipitate during complex formation. 3. Mix 500 μL of the GUS solution into the R9 solution by pipetting repeatedly once more. Incubate solutions for 60 min at 37oC. 4. While the complex solution incubates, prepare the plant tissue. We will be using two types of plant tissue today, mung bean and Duncan grapefruit. Aseptically cut the plant tissue into approximately 1 cm segments (smaller is better) and place into the appropriate wells. 5. Imbibe the segments in each well with 2 mL of MSO at 28oC with 10% DMSO (dilute first) for at least 30 minutes prior to experiment. </p><p>Duncan control Duncan GUS only Duncan GUS + CPP 2 mL MSO + 1 mL PBS 2 mL MSO + .5 mL PBS + .5 mL GUS 2 mL MSO + .5 mL GUS + .5 mL CPP Clean well for rinse Clean well for rinse Clean well for rinse Mung control Mung GUS only Mung GUS + CPP 2 mL MSO + 1 mL PBS 2 mL MSO + .5 mL PBS + .5 mL GUS 2 mL MSO + .5 mL GUS + .5 mL CPP Clean well for rinse Clean well for rinse Clean well for rinse</p><p>6. After incubation, pipet GUS and CPP mixtures into the tissue imbibing solution as shown above. The final CPP concentration is 1 μg of R9/60 μL total volume. (50 μg/ 3mL). 7. Incubate the explants in solution at 28oC for minimum of 60 minutes. The longer the better! 8. During this time media will be provided for rooting flytraps from previous experiment. 9. Remove the tissue from the solution and rinse 5 minutes using 3 mL trypsin solution, 2x. (Pipette into well, allow to sit, remove with pipette, repeat.) This will cleave any exogenous peptide/protein. 10. Move the segments to a clean well below the original wells. 11. Rinse twice using 3 mL sterile water for 5 minutes (same as above). 12. Stain the explants with 3 mL of GUS stain (X-gal solution) overnight. 13. Tomorrow, come in to take observations of your plate.</p>