Joyce: T-tailing pBSKS

Concept

A T-tailed plasmid can be used to rescue DNA that have A-overhangs (as those generated using PCR with Taq polymerase - Taq has terminal transferase activity that adds a single adenosine to the 3'end of its substrate). To make the T-tailed plasmid, cut it first with a blunt cutter, then incubate it with Taq polymerase in the presence of TTP. In the presence of TTP alone, the terminal transferase activity will add a single T to the 3' end of the cut plasmid. This can then be used in a ligation reaction with the PCR products.

Procedure

1. Obtain about 10 ug of pBSKS

2. Cut with a blunt cutting restriction enzyme (like EcoRV)

3. Precipitate the DNA using NaOAc and ethanol

4. T-tail the DNA using:

  10 ug of pBSKS
  10 ul of 10X Taq buffer (no MgCl2)
  0.5 ul 100 mM dTTP 
  6 ul 25 mM MgCl2
  1 ul 5 U/ul Taq
  Sterile dH2O to 100 ul

5. Incubate at 72°C for 2 hours

6. Extract with 1X phenol and 2X ether

7. Precipitate the DNA with NaOAc and ethanol

8. Resuspend in 150 ul of sterile dH₂O

9. Use 1 ul for ligations

Notes

• The pBSKS can be obtained from isolation of a blue colony when cells are transformed with pBSKS. Use a spin column miniprep procedure to isolate the DNA, do a phenol extraction, then precipitate and follow up with RE digestion. The phenol extraction may or may not be necessary, depending on the genotype of the cells used to transform, but if the cells carry a nuclease (endA) this might get carried over and could chew away at your plasmid even before the T-tailing occurs, making screening difficult.
• After the RE digestion and precipitation, it's a good idea to check for complete digestion using either gel electrophoresis (run a sample of uncut for comparison), or transformation. Transformation is preferred because this allows you to see exactly how much undigested plasmid remains (a small number of transformants would indicate that most of the sample has been digested).