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T-vector construction

Simple construction of a T-vector for cloning PCR products

from Marchuk et al (1991), Nucleic Acids Res. 19, 1154

 

This protocol should work on the basis of the fact that given an equimolar amount of dNTPs in a PCR reaction, Taq DNA polymerase I will add an extra (template-independent) A. If the enzyme is supplied with only dTTP, then an extra T will be added.

 

T-vector

Digest the vector to be used in cloning with a blunt-end cutter (such as EcoRV).
Try to achieve at least 150ng/µl.

 

Incubate at 70°C for 2 hours.

The vector can now be run through a spin collumn (eg Qiagen Qiaquick PCR purification collumn) or gene-cleaned. The cleaned vector can now be used in a ligation with a fresh PCR product which has been generated with Taq pol.

 

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