Standard PCR Reaction
Steps for Standard PCR Reaction
1. Design primers. In general, primers should have the following properties:
- Length of 18-24 bases
- 40-60% G/C content
- Start and end with 1-2 G/C pairs
- Melting temperature (Tm) of 50-60°C
- Primer pairs should have a Tm within 5°C of each other
- Primer pairs should not have complementary regions
Tip: Primer3 is an excellent resource for choosing primers.
Tip: If you will be including a restriction site at the 5’ end of your primer, note that a 3-6 base pair spacer should be added in order for the enzyme to cleave efficiently.
2. Set up PCR tubes.
a. Place thin-walled PCR tubes on ice.
b. For a 50 μL reaction, add:
- 2 μL Template DNA (10 ng-500 ng)
- 5 μl 10X Taq buffer with MgCl2
- 1 μl dNTP mix (10 mM each nt)
- 2.5 μL Forward Primer (10 μM stock)
- 2.5 μL Reverse Primer (10 μM stock)
- 0.2 μL Taq DNA Polymerase (5 units/μL)
- 32.8 μL Sterile deionized water (variable)
Tip: If you are doing multiple PCR reactions, save time by creating a “master mix.”
3. PCR: The following is a typical PCR program. The annealing temperature should be 5°C below the primer Tm. The extension step should be 1-2 minutes per kilobase of product, depending on whether you are using a polymerase with proofreading capabilities. See manufacturer’s instructions.
- Step 1: Initial Denaturation for 2 minutes at 95°C
- Step 2: Denature for 1 minute at 95°C
- Step 3: Anneal primers for 30 seconds at 55°C (or 5oC below Tm)
- Step 4: Extend DNA for 2 minutes at 72°C
- Step 5: Repeat steps 2-4 for 25-30 cycles
- Step 6: Final Extension for 10 minutes at 72°C
4. Run 2 μL on a gel to check size and concentration of PCR product.
Reagent List: PCR
- Catalog Number
- NEB # M0267S
- NEB # N0447S
- IDT
Please note that the catalog numbers given in the list above are only examples, and there are many additional companies that supply these reagents.