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^o C
   • Primer pairs should have a Tm within 5^o 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.

    

   1. Place thin-walled PCR tubes on ice.
   2. 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^o 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^o C
   Step 2: Denature for 1 minute at 95^o C
   Step 3: Anneal primers for 30 seconds at 55^o C (or 5^o C below Tm)
   Step 4: Extend DNA for 2 minutes at 72^o C
   Step 5: Repeat steps 2-4 for 25-30 cycles
   Step 6: Final Extension for 10 minutes at 72^o C

    

4. Run 2 μL on a gel to check size and concentration of PCR product.

    

Please note that the catalog numbers given in the list above are only examples, and there are many additional companies that supply these reagents.