# 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
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.