Method for direct cloning a PCR product, by the T-vector technique.
It is relatively easy and cheap to make a vector containing a T overhang. Because Taq polymerase ads an A overhang to PCR products in standard PCR assays, almost any PCR product can be cloned into this vector. The purified T-vector can be stored aliquoted at -70 C for at least one year.
Making the T-vector
1. Digest 5 mg of vector DNA (Bluescript or any other vector) with a restriction enzyme that generates a unique blunt end site, for example EcoRV or Sma I for 2 hours at 37 C.
2. Purify the DNA using the silica method. (I would recommend to run 1/10 th of the material on an agarose gel before purification to check if the cut was working). Elute the DNA from the silica in 45 ml of distilled water.
3. Add 5 mL of 10X PCR buffer, 1 ml of 100 mM dTTP, and 0.5 ml of Taq DNA polymerase. Overlay with 1 drop of mineral oil (if you use a heated lid PCR machine for incubation the mineral oil is not needed). Incubate at 72 C for 2 hours.
4. Purify the T-vector by silica method. Resuspend the prepared T-vector in 100 ml of water, giving a concentration of 100 ng/ml.
Cloning the PCR product
5. Set 3 tubes containing 1.0 ml of either undiluted PCR product*, a 1:10 dilution of the PCR product or distilled water (a control that will indicate the background of vector self-ligation). Add to each tube 1.0 ml of T-vector, 2 ml 5X ligation buffer (with DTT and ATP), 6.5 ml of distilled water and 0.5 ml of T4 DNA ligase. Incubate overnight at 16 C.
Use the ligated product for standard transformation assay. It is recommended to use blue white selection. *We got better yield if the PCR product was purified using the silica method (70-90% of the clones contained the PCR products) before ligation. Generally, between 30-70 clones are generated per plate.
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