User:Melissa Novy/Notebook/CHEM-571/2012/10/16

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Objectives

  • 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)
  V1=204.08 μL


  • Procedure
    1. Add 1000 μL of deionized water to 0.49 mg of ADA K110A (r).
    2. 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.
    3. 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.
    4. 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
Component Volume [μL]
Distilled water40.6
10X cloned Pfu buffer5
25 mM dNTPs0.4
100 ng/μL DNA template1
Forward primer (K110 f)1
Reverse primer (K110A r)1
2.5 U/μL PfuTurbo DNA polymerase1


  • Temperature Cycling Settings for Thermocycler
    1. Heat for 2 min at 95°C.
    2. Heat for 30 s at 95°C.
    3. Heat for 30 s at 57°C.
    4. Heat for 8 min at 72°C.
    5. Repeat steps 2 through 4 30 times.
    6. Heat for 10 min at 72°C.
    7. 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.



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