Realizing the Rookie Mistake

• only some enzymes can cut right at a restriction site. some need 1, 2, 3+ basepairs on the ends
• https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

Checking What We Have for Polyermase

Primers Stock (our stock so we can use it again later)

• they arrive dry?
• 10x of nuclease free water of nano/mole (MBGW = molecular biology grade water = nuclease free h20)
• add right to primer container
• future storage in -20
• https://toptipbio.com/how-to-dilute-new-pcr-primers/
  • “For example, if a primer states 19.4 nmoles, then add 194 μL of PCR-grade water.”
• FYI: put it in the -80ºC

PCR?

• how much of the primers (called working stock)???????
  • mentioned in company’s documentation. likely 1:10 dilution (1µL of primer, 9µL of water)
    • side note, i wish instead of people saying 1 to 10 dilution, we said 1 of 10 dilution
• how much of the master mix?????
  • mentioned in company’s documentation