PCR Protocol(per Adam 08/12/04) 【University of Chicago】

PCR (polymerase chain reaction) is a sensitive procedure that amplifies specific regions of DNA. A forward and reverse primer flank a specific portion of DNA, throughout many cycles of PCR a single copy of DNA can be amplified exponentially.

1. Sign up for the thermal cycler (PCR machine) to be used ahead of time.

2. Dilute DNA sample(s) 1:10 or 1:25 (depending on original concentration.)

3. Add DNa to PCR plate (5µL per reaction).

4. Start the desired PCR program on the thermal cycler of use. Once the program is started, PAUSE it to allow the machine to heat up to 94-95°C.

5. Prepare PCR cocktail per the following recipe:

DNA Template MgSO4(25mM)* 10X PCR Buffer dNTPs (2.5 mM each) Forward Primer (20µM) Reverse Primer (20µM) ddH2O* Taq*** (5 units/µL) TOTAL VOLUME

w/MgSO4 5.0µL 5.0µL 2.5µL 2.0µL 0.3µL 0.3µL 9.8µL 0.1µL 25µL

w/o MgSO4 5.0µL ------- 2.5µL 2.0µL 0.3µL 0.3µL 14.8µL 0.1µL 25µL

*Addition of Mg2 stabilizes denatured DNA, it can improve the yield of PCR products (specific and nonspecific). It is a variable to conider manipulating if your PCR efforts are unsuccesful.
***It is important to add the Taq to the cocktail last.

6. Add cocktail to samples.

7. Begin PCR by putting plate in the thermal cycler (once it has reached 94-95°C) and pressing PAUSE again, to allow the reaction to proceed.

8. Once completed, mix 5µL PCR products and 1µL 6X loading dye to sample prior to gel analysis.