- To run PCR on ADA DNA primer
- To make Au/BSA solutions
- Calculations were made to make 100ng/μL solution of ADA E34Af primer.
100ng/μL *(g/10^9 ng)* (mol/15076.9g) * (10^6 μL/L) = 6.633E-6M
32.8E-9 mol * (L/6.633E-6 mol) = 0.004945L
- Because no more than 1 mL of water could be added to the primer, the volume added was 1mL, making a 4.95x reduction of volume and, therefore, a 4.95x increase in concentration.
- So, a dilution was made to get a concentration of 100ng/μL.
(4.945) v1= (1)(0.001L)
v1= 2.022E-4 L
1000μL-202.2μL=797.8μL water added
- 40.6μL distilled water was added
- 5.0μL 10x cloned Pfu reaction buffer was added
- 0.4μL (25mM each dNTP) dNTPs were added
- 1.0μL 100ng/μL DNA template was added
- 1.0μL 100ng/μL Primer #1 (ADA E34A f) was added
- 1.0μL 100ng/μL Primer #2 (ADA E34A r) was added
- 1.0μL (2.5U/μL) PfuTurbo DNA Polymerase was added [immediately before placing in PCR chamber.
- The Temperature Cycle for the PCR is as follows:
- Heat for 2 min at 95°C.
- Heat for 30 s at 95°C.
- Heat for 30 s at 57°C.
- Heat for 8 min at 72°C.
- Repeat steps 2 through 4 30 times.
- Heat for 10 min at 72°C.
- Chill at 0°C until needed.
- In addition to the PCR mutation, Au/BSA solutions were made.
- 0.0398g BSA was added to 20mL water
- Please refer to 2012/10/03| Melissa's entry from last week for information, calculations, and measurements on the Au/BSA stock solution made. 80μL of this stock solution was used to have 0.00000208 mol of HAuCl4.
- The following table was used to determine volumes of addition 14.9μM BSA added and water added.