Running your first PCR
It's best to start small with your first PCR. The best way to run a test PCR is to just run a standard curve.
To set up a standard curve, follow these steps:
- Dilute your cDNA 1:10. Assemble a random mix of six or so of your samples to a final volume of 10 uL.
- Set up 6 tubes with 10 uL of water in each.
- Add 10 uL of your mixture to tube 1. Pipet up and down to mix, and then take 10 uL of tube 1 into tube 2.
- Repeat until you get to tube 5.
- Leave tube 6 alone, it is the water blank.
- Add 10 uL from each of these tubes into wells of a 96 well PCR plate. Prepare reaction mix using the master mix 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.