TOPO TA Subcloning (Invitrogen)

Takes about 2 hours

Preparation phase

1. Spread 80 ul of 20 mg/ml X-gal (stored at -20) on 2 Amp plates/transformation.
2. Thaw TOPO-TA 1-shot competent cells (stored at -80) on ice.
3. Equilibrate a water bath to 42 ¡C.

Reaction phase

1. . Add to a 0.5 ml Eppendorf tube:
   • 4 ul PCR reaction without purification or quantification.
   • 1 ul salt solution from kit
   • 1 ul vector solution from kit
2. Mix gently and incubate for 5 minutes at room temperature

Transformation phase

1. Add 2 ul to TOPO TA 1 shot cells on ice, mix gently
2. Incubate 5 - 30 minutes on ice
3. heat shock for 30 seconds at 42 C and put back on ice.

Grow out and plating

1. Add 250 ul of SOC medium
2. Grow out at 37 C for 1 hour shaking at 250 rpm.
3. Plate 10 ul and 50 ul on separate plates, incubate at 37 C until colonies come up.

Screening

1. Culture 4 white colonies/transformation in 5 ml LB containing Amp.
2. Screen by mini prep and agarose gel.

note:*Carbenicillin may also be used. Amp and Kan or Carb and Kan may be used in conjunction. The TOPO TA kit for cos cell expression uses a different vector with different antibiotic resistance.