This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
T-A cloning Vectors

T-A cloning Vectors


[Papers To Read]



Search for

Prediction of Genetic Interactions (version 140) Advanced Search | Gene Pair Info

Gene name:


Use this page to upload/download files






[Web-based Email]

[Web Proxy Service]

Chin-Sang Lab Queen's Upload/Download Page

Locations of visitors to this page

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:


1. Digest 5 ug pBluescript II with EcoRV (blunt cutter).


2. Heat kill the enzyme or gel purify using Qiagen column elute in 50ul EB.


3. 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.


4. 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:

5.  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.

For example:

PCR insert                    10ul

T-Vector                      5ul

5X Ligase buffer           4ul

T4 Ligase                     1ul


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.



Ref: Marchuk, D., et al. (1991) Nucleic Acids Res. 19, 1154