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Objective
Today we are going to conduct two different experiments:
- We are going to synthesize some Gold NPs again (tube 1) and we are going to investigate how the protein aggregate forms (tube 2; we are using an acidic solution to test if this property is responsible for the formation of the aggregate; the HAuCl4 is also an acidic solution).
- We are going to conduct a PCR again to produce some mutate proteins.
Protocol
- Tube 1 (in this specific order): add 1ml of BSA stock solution (15.5µM) + 1ml of HAuCl4 stock solution (2.9mM) + 8ml of water
- Tube 2 (in this specific order): add 1ml of HCl stock solution (3m) + 1ml of BSA stock solution (15.5µM) + 8ml of water
- Leave in the oven for 2 hours; remove every 30 minutes and leave at room temperature for 10 minutes
- PCR: we are using the same protocol as on the 20th of September but we are using different primers
- Add 5µL of 10X Pfu Buffer + 1µL of each primer + 1µL of DNA sample + 1µL of dNTP mix + 40µL of water + 1µL of Pfu Turbo + 50µL of Wax solution
- Place the tube in the thermocycler whose temperature cycling program is:
- 30 sec at 95°C
- 30 sec at 60°C
- 7 min at 72°C
- Repeat these three steps 19 times
- 10 min at 72°C
- Leave overnight at 4°C
The forward primer is 5'TACGACGATGACGATAAGTGTCGATGGGGATCCGAATTC3' and the reverse primer is 5'GAATTCGGATCCCCATCGACACTTATCGTCATCGTCGTA3'
Results
In tube 1, we obtained an incolore solution with a black/dark purple aggregate.
In tube 2, nothing happened, the solution remained incolore and no fibers or filaments was formed.
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