Build-a-Gene Session 1

Template-dependent PCR

The goal of template-dependent Polymerase Chain Reaction (PCR) is to exponentially amplify a DNA sequence so that we produce many copies of that one DNA segment. In this case, we will be amplifying the vector that will carry the Emerald GFP gene and the promoter that will allow the Emerald GFP gene to be expressed and produce protein in the cells.A good introduction to PCR is here:[1]

Reaction Setup

• Keep the master mix on ice AT ALL TIMES!
• When each reagent has thawed, mix briefly by pipetting up and down several times.

1. Create your reaction mixes by combining all reagents listed below into a PCR tube.

Reagent	vector PCR (pSB1C3)	promoter PCR (J04500)	!Control PCR
PCR master mix (contains buffer, enzyme and nucleotides)	10 ul	10 ul	10 ul
Primers	5 ul vector primers	5 ul of each of the promoter primers	Add 5 ul of each of the promoter primers
DNA template	Add 5 ul of the vector template	Add 5 ul of the promoter template	Add 5 ul of water
Total	25 ul	25 ul	25 ul

2. Vortex briefly so that your reaction mix is completely mixed. Cap your tubes and make sure that they are sealed tightly so that the liquid will not evaporate. Place your tubes in the PCR machine-the vector PCR will go in the block on one side of the machine and the promoter and control PCR will go on the other side. Record which wells contain your tubes, making sure that you have recorded which sample (the promoter PCR or the control PCR) is in each position.

Reaction Conditions:

95°C, 3 minutes

30 cycles:

       95°C, 20 seconds        
       55°C, 20 seconds
       68°C, 2 minute

68°C, 5 minutes