Testing your Primers

Before running an actual PCR with your samples, you must test your primers and samples to make sure they are adequate. 

1.  Test your primers using a standard curve generated from your samples, and a water blank.  A standard curve is simply a 1:2 serial dilution of a random set of your samples.  Five dilutions is enough. 

2.  Run a dissociation curve to indirectly test for multiple products. 

3.  Run a RNA sample to test for genomic DNA contamination. 

4.  Run some product on a gel and make sure your product(s) are the correct size.  (some labs don't do this). 

5.  Subclone your product into a T vector (such as the pGem T-Easy kit from Promega), and sequence ten products to make sure that the product is correct.  This is the only way you can be absolutely sure that you are amplifying the correct product, and that a second product isn't the same size as your presumed target. (most labs don't do this).  

Other comments: 

TaqMan primers eliminate the need for testing most amplifications by PCR.  Because a third probe is involved, spurious amplifications are not detected by this method, even if they do occur.