User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/10/16

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Objective

  • To run PCR on ADA DNA primer
  • To make Au/BSA solutions

Description

  • 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.

m1v1=m2v2

(4.945) v1= (1)(0.001L) v1= 2.022E-4 L 1000μL-202.2μL=797.8μL water added

  1. 40.6μL distilled water was added
  2. 5.0μL 10x cloned Pfu reaction buffer was added
  3. 0.4μL (25mM each dNTP) dNTPs were added
  4. 1.0μL 100ng/μL DNA template was added
  5. 1.0μL 100ng/μL Primer #1 (ADA E34A f) was added
  6. 1.0μL 100ng/μL Primer #2 (ADA E34A r) was added
  7. 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
      • 14.9μM BSA solution
    • 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.

Image:BSA_solution_table_20121016.png



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