PCR Protocols

Depending on the DNA polymerase you use, final concentration of each reagent will vary. These are the standard volumes and concentrations taking into account a 50ul reaction:

1. In PCR tubes of 200 μl:
   1. Add 38 μl sterile water.
   2. Add 2 μl of forward primer (10 μM).
   3. Add 2 μl of reverse primer (10 μM).
   4. Add 1 μl of dNTPs (50 μM).
   5. Add 5 μl of reaction buffer containing MgCl2 (10X).
   6. Add 1 μl of DNA template (100 ng/μl).
   7. Add 1 μl of DNA polymerase (0.5 U/μl).

• Note: If you are willing to do a significant number of PCR reactions, it is recommended to prepare a reaction mix, excluding the reagents that will be different from experiment to experiment (usually the DNA template or the set of primers).

2. Pipette gently the reaction mixture to allow good homogenization.
3. Short spin in a centrifuge is recommended.
4. Make sure the caps of PCR tubes are closed.
5. Insert the tube in the thermocycler.

Tip: Set up the PCR settings, such as temperature, time and cycles before your PCR reaction is ready. See the diagram below for reference of typical PCR reaction settings.

Note: Always pay attention to the guidelines of the DNA polymerase, since denaturation, annealing, and extension temperature and time highly depend on it. In the thermocycler, you should set up these steps subsequently (refer to diagram above):

1. An initial step of DNA denaturation at 94°C to 98°C for 3 to 5 min depending on the optimal temperature of the DNA polymerase
2. Denaturation temperature (the same used in the step before) for 30 seconds
3. Annealing temperature taking into account the Tm of the primers for 30 seconds
4. Extension temperature at 72°C and taking into account the size of the fragment to be amplified

These steps should be repeated for 25 to 35 rounds (cycles). A final step of extension is required to allow all the PCR products to be correctly synthesized, usually at 72°C for 10 min. Finally, the temperature should be reduced to 4°C to store the PCR product.

1. Load the 6 μl mixture in an agarose gel 1%.
2. Load 5 μl of DNA marker in the same gel.
3. Use an UV transilluminator to visualize the PCR product in the agarose gel.

Note: In order to avoid staining with ethidium bromide, you can use Midori or Red Safe pre stained gels, which are less toxic compounds. Use a DNA marker to compare the correct size of the PCR product.

1. In a 200 μl PCR tube:
1. Add 2 μg of total RNA.
2. Add 1 ul of oligo dt (50μM).
3. Add 1 μl of dNTP (10mM).
4. Add DEPC-treated water up to 10 μl.

2. Incubate at 65 °C for 5 min and then keep the tubes on ice for 1 min.
3. Add 2 ul of RT buffer (10X) .
4. Add 4 μl of MgCl₂.
5. Add 2 μl of DTT (0.1 M).
6. Add 1 ul RNase inhibitor (40 U/μl).
7. Add 1 ul of reverse transcriptase (200 U/μl).
8. Incubate at 50°C for 60 min, followed by 85°C for 5 min.
9. Afterwards, keep the tube on ice.
10. Add 1 μl RNase H.
11. Incubate for 30 min at 37°C.
12. Store the PCR tube at -20°C.
13. For RNA quantification and quality, use a NanoDrop equipment to measure the RNA (260 nm), protein, and salt concentrations.

For each cDNA gene, prepare the following reaction mix in 200 μl PCR tubes for a final volume of 50 μl:

• Add 25 μl of SYBR Green Mix (2x).
• Add 1 μl cDNA from the previous cDNA sample.
• Add 1 μl Forward Primer (10 mM).
• Add 1 μl Reverse Primer (10 mM).
• Add 22.5 μl DEPC Water.
• Insert the tubes in the thermocycler.

When the PCR is finished, perform quantification analysis by comparative Ct method. The Ct method compares the Ct values of the samples with standards samples. The Ct values of the controls and the samples are normalized using the Ct values obtained for housekeeping genes. To validate the Ct calculation, it is required that the amplification efficiencies of the target should be close to endogenous. This can be accessed through the utilization of different template dilutions.