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.

Department of Microbiology
School of Medicine
University of Washington
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