Running your First PCR

It's best to start small with your first PCR.  You'll first want to test your primers to make sure that they work. 

First, run a test PCR with your primers.  You can do this with a few of your samples, and by running a standard curve.  A standard curve that works shows you that both your samples and your primers are good. 

To set up a standard curve, follow these steps:

1. Dilute your cDNA 1:10.  Assemble a random mixture of samples. 
2. Set up 6 tubes with 10 uL of water in each.
3. Add 10 uL of your random mixture to tube 1.  Mix, and add 10 uL to tube 2.
4. Repeat to tube 5. 
5. Leave tube 6 alone - it is the water blank. 
6. Add these six tubes to wells, and add reaction mix using the Recipe Template. 

If you have more than one primer to test, repeat this for each primer. 

When loading your plate into the real time PCR machine for the first time, have someone help you set up the machine.  The program needs to be told which dye it is reading.  If you don't select one, none is read.  You then lose your data.  Also, be sure to add the dissociation protocol to the program.  This will help show whether your primers are good.