Steps for Standard PCR Reaction

Steps for Standard PCR Reaction

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

o Length of 18-24 bases

o 40-60% G/C content

o Start and end with 1-2 G/C pairs

o Melting temperature (Tm) of 50-60oC

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

o 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.

o Place thin-walled PCR tubes on ice.

o For a 50 μL reaction, add:

o Tip: If you are doing multiple PCR reactions, save time by creating a "master mix."

1. PCR: The following is a typical PCR program. The annealing temperature should be 5oC 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 95oC
Step 2: Denature for 1 minute at 95oC
Step 3: Anneal primers for 30 seconds at 55oC (or 5oC below Tm)
Step 4: Extend DNA for 2 minutes at 72oC
Step 5: Repeat steps 2-4 for 25-30 cycles
Step 6: Final Extension for 10 minutes at 72oC

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