# **Polymerase Chain Reaction ( PCR ) for gDNA**
Usage amount per sample:
| Material ※ | Concentration | Addition per sample | Location |
| :----------- | :---: | :---: | :---: |
| gDNA † | 10 ng / uL | 4 μL | Usually -20°C |
| F-primer | | 0.4 μL | -20°C ( 2-3-4 ) |
| R-primer | | 0.4 μL | -20°C ( 2-3-4 ) |
| 2X-Taq (orange lid) | | 5 μL | -20°C ( 4-3-4 ) |
| Ambion water | | 0.2 μL | RNA bench |
| Total volume: | | 10 μL | place on ice |
※ All materials should be placed on ice.
† gDNA : The gDNA stock solution of all samples should be standardized to the same concentration, for example 10 ng/μL.
# Procedure
1. Prepare the total amount of the required working solution ( F-primer, R-primer, 2X-Taq );
- For example, if we had 10 samples, then me make the working solution by adding 4 μL F-primer, 4 μL R-primer, and 50 μL 2X-Taq into a new 200 μL PCR tube.
2. Dispense the working solution into a new 200 μL PCR tubes ( 5.8 μL for each sample );
3. Lastly, add the sample gDNA stock solution;
4. Close the lid tightly, and swipe the tube gently with finger several time to mix the solution well;
5. Slightly centrifuge to bring down the solution;
6. Put into the PCR machine ( the machine should be warmed up around 2 mins before use ).
# Reaction curve
| Process | Temperature (°C) | Time | Cycle |
| :----------: | :---: | :---: | :---: |
| Initial denaturation | 95 | 1 min | -- |
| Denaturation | 95 | 15 secs | 30 |
| Annealing † | 56 | 15 secs | 30 |
| Extension | 72 | 1 min | 30 |
| Complete extension | 72 | 5 min | -- |
| PCR product storage | 4 | ∞ | -- |
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† Annealing temperature depends on primers.
Done !!!
If you encounter any issues with the protocol, please report them at:
[https://github.com/P10911004-NPUST/jklai/issues](https://github.com/P10911004-NPUST/jklai/issues).