Protocols >

Whole Cell PCR

Adapted from D. Kirkpatrick (4/15/98)

• Put 6µ l of dH₂O into a 0.2 ml eppendorf tube.
• Transfer cells from colony on plate to the tube using a toothpick.
• Small amount of cells is best — water should be translucent.
• Use a brand new toothpick, discard after use.
• Heat tube at 94^oC for 6 min. in PCR machine.
• Place tube in -80^oC freezer for 5 min. (minimum), can go overnight.
• Add 19µ l PCR mix:
  • dH₂O 3.25µ l
  • 10X buffer 2.5
  • 25mM MgCl 1.5
  • 1µ M Primer 1 5
  • 1µ M Primer 2 5
  • 10mM dNTP 0.5
  • Taq 0.25
  • TOTAL 19µ l
• Mix by pipetting.
• Put in PCR machine, run program <Eric> folder, Genomic
  • 94^oC 3min.
  • 94^oC 30sec.
  • 55^oC 30sec. 40 cycles
  • 72^oC 3min.