StandardPCRreaction

Steps for Standard PCR Reaction

1. Design primers. In general, primers should have the following properties:

   
   

   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.

   
   

• 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^oC

• Primer pairs should have a Tm within 5^oC of each other

• Primer pairs should not have complementary regions

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^oC 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^oC
   Step 2: Denature for 1 minute at 95^oC
   Step 3: Anneal primers for 30 seconds at 55^oC (or 5^oC below Tm)
   Step 4: Extend DNA for 2 minutes at 72^oC
   Step 5: Repeat steps 2-4 for 25-30 cycles
   Step 6: Final Extension for 10 minutes at 72^oC

   
   

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

   
   

Reagent List: PCR

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