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Cloning of PCR products

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. Although this procedure is simple and rapid, number of colonies obtained after transformation of E. coli is usually smaller than that obtained by standard cloning techniques. If you need large number of independent clones, I recommend to carry out standard procedure described here.

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).
**) 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, SmaI or EcoRV, depending on the sequence of the PCR products.

2. Incubate for 4 hours at 22 degrees.

3. (Optional) Incubate for 10 minutes at 65 degrees to inactivate the enzymes. Add the restriction enzyme (2 units) . Incubate for 30 minutes. This step reduces the ratio of intact plasmid in the reaction mixture.

4. Transform E. coli using 0.5 ul of the reaction mixture. Plate the transformed cultures on plates containing IPTG and X-gal. Check the length of the inserts in the plasmids by PCR. Typically, 10-80% of white colonies contain recombinant plasmids.

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

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