TOPO T/A Cloning of PCR Products
Addition of 3'A-overhangs post amplification
Add 0.7 - 1.0 U of Taq polymerase (Biolase) to PCR product.
Mix well. Incubate at 72 Â°C for 8-10 minutes. Use immediately.
Modified Topo T/A cloning protocol
Spread each Carb. plate with 40 Âµl of 40mg/ml X-gal.
Pre-warm plates in warm room.
Dilute PCR product 1 in 4 with dH2O.
Topo T/A vector mix 0.25 Âµl
PCR product dilution 1.00 Âµl
dH20 1.25 Âµl
Incubate 5 minutes at room temperature, and then put on ice.
Top10 cells should be thawed GENTLY and placed on ice; keep everything cold.
Ligation 1.5 Âµl
0.25ul Î²-ME 1.0 Âµl
Top10 cells 10.0 Âµl
Stir gently with pipette tip.
Chill on ice for 15 minutes.
Heat shock in 42Â°C water bath for 30 seconds.
Chill on ice for 2 minutes.
Add 125 Âµl SOC-media and incubate -shaking- at 37Â°C for 1 hr.
Plate the entire transformation on LB-carb + X-Gal plates.
Place plates in warm room overnight.
When cloning a 750 bp product, this procedure consistently gives 100-200 colonies per plate.