- Prepare ADA primers for polymerase chain reaction (PCR).
- Conduct PCR on the primers.
- Follow protocol on 2012/10/03, using the same mixed Au/BSA stock solution, to make 10 mL of Au-BSA solutions for AAS analysis.
Adenosine Deaminase Mutations
- Assigned primer: ADA K110A (r)
- Issue number: 87635083
- Sequence: 5' - CCA TTA AAA CAA CAC CTT CTT TAT ATG
- MW: 16401.7
- Tm: 65.5°C
- 29.9 nanomoles = 0.49 mg
- The volume of water necessary to produce 100 ng of primer per μL of solution is the following:
0.49 mg = 490 × 103 ng
[(490 × 103 ng)×(1 μL)]/(100 ng) = 4900 μL
490 × 103 ng/4900 μL = 100 ng/μL
Note that 490 × 103 ng/1000 μL = 490 ng/μL, such that (490 ng/μL)×(V1)=(100 ng/μL)×(1000 μL)
- Add 1000 μL of deionized water to 0.49 mg of ADA K110A (r).
- Add 204.08 μL of this solution into a centrifuge tube and dilute to 1 mL with autoclaved water to give a solution of 100 ng of primer per μL of solution.
- Two reaction mixtures of 50 μL each were prepared by adding the following components into each of two microcentrifuge tubes in the order listed in the table below.
- Upon the addition of DNA polymerase, the microcentrifuge tubes were capped and placed in a thermocycler set to the program below.
- Volumes of Reaction Components Added to PCR of ADA Reaction Solution
| Distilled water||40.6
| 10X cloned Pfu buffer||5
| 25 mM dNTPs||0.4
| 100 ng/μL DNA template||1
| Forward primer (K110 f)||1
| Reverse primer (K110A r)||1
| 2.5 U/μL PfuTurbo DNA polymerase||1
- Temperature Cycling Settings for Thermocycler
- 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.
Au/BSA Solutions for AAS
- Please refer to the protocol on 2012/10/03. Refer to Dhea Patel's entry for calculations and volumes of components added to the Au/BSA solutions.
- The concentration of BSA stock solution used was 14.9 μM in water. Note that 20 mL of this solution was made and stored in the refrigerator at 4°C.
- 0.0398 g of BSA was added to 20 mL of water in a Falcon tube to give a solution of 14.9 μM BSA.
- 10 mL of solutions at the following Au to BSA ratios were made: 60, 80, 100, 120, 128, 130, 132, 133, 134, 136, 138, 140, 160, and 170.