"

From 2010.igem.org

Johan

Colony PCR

• Another colony PCR on the second transformation of TAT CCP as the first one didn't show many colonies and none of the colonies showed any insert in the vector.

• Colony #1-36 +insert, colony #42-43 -insert.

• PCR reaction mix
  • 0,5 µl Pfu polymerase
  • 0,5 µl 10 mM dNTPs
  • 2 µl 10 µm f.primer (VF2)
  • 2 µl 10 µm r.primer (VR)
  • 2,5 µl Pfu buffer 10x
  • 17,5 µl H2O

• PCR program
  • 98 °C - 2 min
  • 35 cycles of
    • 98 °C - 10 sec
    • 55 °C - 15 sec
    • 72 °C - 45 sec
  • 72 °C - 5 min
  • 4°C - ∞

Gel electrophoresis

A gel electrophoresis was performed on the PCR products.

Lane 1 & 40: FastRuler DNA Ladder low range, lane 2-37: +insert, lane 38-39: -insert.

The gel didn't turn out good, probably I used too much DNA template.