Difference between revisions of "TOPO Cloning"

Latest revision as of 00:45, 2 May 2009

1. PCR desired insert using Pfu polymerase. Add 1 uL of Taq and mix. Incubate 10min at 72C to add A to 3’ ends. Use immediately
2. If PCR product is a single band, no need to purify further. If several bands, gel purify.
3. Combine 4 uL PCR product, 1 uL Salt Solution, and 1 uL TOPO vector. Mix gently
4. Incubate at room temperature for 5 min, place on ice.
5. Thaw tube of OneShot cells on ice
6. Add 2 uL reaction and incubate on ice 5-30 min
7. Heat shock 30s at 42C without shaking
8. Place on ice for 2 min
9. Add 250 uL of room temperature SOC media
10. Incubate at 37C for 1h
11. Plate 50 uL on plate and grow overnight