Cloning of PCR products (One-tube reaction)

In the following protocol, recombinant plasmids can be generated in a single reaction tube from an intact plasmid and unpolished PCR products. .

1. Mix the following solutions.

Intact plasmid (50ng/ul)
PCR product (gel-purified)
10x Universal KGB buffer*
dNTP mixture (2.0-2.5mM each)***
restriction enzyme**
T4 DNA polymerase***
T4 DNA ligase
1 ul
0.05 - 0.5 pmol
2 ul
2 ul
1 ul
2 units
1 units
3 units
Adjust to make total reaction volume of 20 ul

*) 10x Universal KGB buffer contains 1 M potassium acetate, 0.25 M Tris-acetate (pH 7.6), 0.1 M magnesium acetate, 5 mM 2-mercaptoethanol, and BSA (0.1 mg/ml). This buffer can be replaced by either 10x NEBuffer 4 (+BSA) (New England Biolabs) or 10x buffer T (+BSA) (Takara).
**) The restriction enzyme should cut the vector plasmid at a single site generating blunt ends. Also, there should not be the restriction site in the PCR product. I usually use HincII or SmaI depending on the sequence of the PCR products.
***) The four dNTPs and T4 DNA polymerase are added for polishing the termini of PCR products that may have 3'-protruding ends generated by the template-independent terminal deoxynucleotidyl transferase activity of Taq DNA polymerase. If the DNA polymerase used in PCR had 3'-exonuclease activity for proofreading, dNTPs and T4 DNA polymerase should be omitted from this reaction since termini of PCR products are already polished during PCR by the action of the 3'-exonuclease activity.

2. Incubate for 4 hours at 22 degrees or overnight at 16 degrees.

3. (Optional) Incubate for 10 minutes at 65 degrees to inactivate the enzymes. Add the restriction enzyme (2 units) . Incubate for 0.5 - 16 hour. This step reduces the ratio of intact plasmid in the reaction mixture.

4. Transform E. coli using 2 ul of the reaction mixture. Spread the transformed cultures on agar plates containing an appropriate antibiotic.

Reference: Molecular Cloning, 3rd edition (Sambrook and Russell eds.), CSHL Press, 2001.

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