Prerequisite Protocols

• PCR Primer Design

Materials

• Taq 5x Master Mix
• Forward PCR Primer Working Stock 10uM
• Reverse PCR Primer Working Stock 10uM
• Sterile Distilled Water
• Template DNA (miniprep, genomic isolate, etc)
• Sterile 0.2mL PCR Tubes
• Sterile 1.5mL Tubes
• Pipette (2.5uL, 20uL, 200uL)
• Purple Loading Dye 6x

Equipment

• PCR Machine
• Thermoshaker set to 37C 1000rpm
• Vortexer
• Ice Bucket with Ice or Cooling Block for 0.2mL and 1.5mL Tubes

Step By Step

1. Decide how many templates you have or wish to screen and write down this number. Obtain that many sterile 1.5mL tubes. Add 198uL of sterile distilled water to each of those tubes.
2. To each tube, add 2uL of your desired template DNA. Label the tubes and do not cross contaminate. This is a typical dilution for minipreps as too much template can be bad. Do not add more than 100ng of template DNA for PCR runs. Vortex the tubes and place on ice. These will act as your template for the rest of the experiment.