Joyce: T-tailing pBSKS

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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 11 ug of pBSKS

2. Cut with a blunt cutting restriction enzyme (like EcoRV) in a 100 ul volume

3. Remove a 10 ul aliquot to use as a control

4. Precipitate the remaining 90 ul (~10 ug) using NaOAc and ethanol

5. 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. Perform 2 sets of transformation controls as follows:

  pBSKS-digested + ligase --> many colonies
  pBSKS-digested - ligase --> very few (indicates complete digestion was successful by showing  
                                        low background of undigested pBSKS)
  T-tailed + ligase       --> very few (shows amount of digested pBSKS that doesn't have a T-tail)
  T-tailed - ligase       --> close to 0 (shows amount of undigested pBSKS in sample)


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 take a 10 ul aliquot and run it on a gel along with uncut plasmid to confirm complete digestion (otherwise, add more RE and incubate again). BETTER YET, take this 10 ul (about 100 ng), and transform cells to show complete background. The problem with doing just electrophoresis is that you might have undigested plasmid that is co-migrating with your digested sample, so it may look like everything is completely digested, but it could be tainted with undigested DNA with some degree of supercoiling. You should do this before moving onto the T-tailing, as you might find a high degree of undigested pBSKS which would require further digestion.
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