Addition of 3'A-overhangs post amplification

1. Add 0.7 - 1.0 U of Taq polymerase (Biolase) to PCR product.

2. Mix well. Incubate at 72 °C for 8-10 minutes. Use immediately.

Modified Topo T/A cloning protocol

Preparation:

1. Spread each Carb. plate with 40 µl of 40mg/ml X-gal.

2. Pre-warm plates in warm room.

Ligation:

1. Dilute PCR product 1 in 4 with dH₂O.

2. Combine:
   Topo T/A vector mix 0.25 µl
   PCR product dilution 1.00 µl
   dH₂0 1.25 µl

3. Incubate 5 minutes at room temperature, and then put on ice.

Transformation:

1. Top10 cells should be thawed GENTLY and placed on ice; keep everything cold.

2. Combine:
   Ligation 1.5 µl
   0.25ul β-ME 1.0 µl
   Top10 cells 10.0 µl

3. Stir gently with pipette tip.

4. Chill on ice for 15 minutes.

5. Heat shock in 42°C water bath for 30 seconds.

6. Chill on ice for 2 minutes.

7. Add 125 µl SOC-media and incubate -shaking- at 37°C for 1 hr.

8. Plate the entire transformation on LB-carb + X-Gal plates.

9. Place plates in warm room overnight.

10. When cloning a 750 bp product, this procedure consistently gives 100-200 colonies per plate.