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T-A cloning Vectors

T-A cloning Vectors

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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