Vector for TA cloning

Virtualab Protocols (Chang Bioscience)
PCR products using Taq DNA polymerase may have an extra nucleotide, most likely A, at their 3'-ends because of the transferase activity of Taq DNA polymerase. Other PCR polymerases may not have the transferase activity. PCR products with a 3' A can be ligated to a vector with 3' T.

1. To make a vector with 3' T (T-Vector), digest 5 mg vector DNA with an restriction enzyme that produces blunt ends. Check with agarose gel electrophoresis to make sure the digestion is complete. Add a little fresh enzyme and continue digestion if necessary.

2. After completion of the digestion, cool the digestion mix on ice. Remove protein with phenol:chloroform:isoamyl alchol (25:24:1) extraction, and precipitate DNA with ethanol.

3. After wash and dry, resuspend the pellet in 172 ml of ddH2O. Make the following mix:

Digested DNA in ddH2O (5 mg) 172 ml
10X PCR Buffer 20 ml
10 mM dTTP 4 ml
Taq DNA polymerase (10 units) 4 ml

4. Incubate at 72 C for 1 hour to make the T-Vector. Incubate either in a 72 C heat block or program PCR thermocycler to run at 72 C for 1 hour.

5. Add 1 ml to a ligation mix to test for self-ligation. Transform E. Coli cells with 1 ml of unligated T-Vector, and self-ligation products. Few clonies should form for both.

6. For cloning of PCR products, ligate 1-5 ml of PCR products to 1 ml of T-Vector.