T-A cloning Vectors¶
Contributed by Ian Chin-Sang, Queens University, ON, Canada
A method for direct cloning a PCR product, by the T-vector technique. This is cheap and easy way to clone PCR products with A 3’ overhangs.
- Making the T-vector:
Digest 5 ug pBluescript II with EcoRV (blunt cutter).
- Heat kill the enzyme or gel purify using Qiagen column elute in 50ul EB.
- Add 10 ul of 10X PCR buffer, 2 ul of 100 mM dTTP, 37.0ul of distilled water and 1.0 ul of Taq DNA polymerase. Incubate at 72 C for 2 hours.
- Purify the T-vector by phenol/chloroform extraction and ethanol precipitation or purify over a Qiagen column. Resuspend/elute the prepared T-vector in 100 ul of water or TE. We use about 2- 5ul in a ligation reaction.
- Cloning the PCR product:
Purify the PCR product over a Qiagen column (Gel purify if necessary). Elute in 50 ul EB. Use 5-10 ul of this PCR product in a ligation reaction e.g.
- 10ul PCR insert
- 5ul T-Vector
- 4ul 5X Ligase buffer
- 1ul T4 Ligase
- Ligate 15min RT(rapid ligation kit) to overnight (15C).
- Transform into E. coli (XL1Blue).
- Plate on Amp (75ug/ml) X-gal (spread 60ul of 2% solution on plate) Tet (15ug/ml) IPTG ( 0.1-1mM)
- Pick white colonies to prep.
Marchuk D, Drumm M, Saulino A, Collins FS Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res (1991) pmid:2020552