Joyce: T-tailing pBSKS: Difference between revisions
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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]] | [[Procedure]] | ||
Revision as of 10:37, 12 September 2009
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.
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
- This isn't necessary with pure plasmid but if you extract the plasmid from a cell line that doesn't have a mutation in endA (or some other nuclease), then you should treat it with phenol to remove the nuclease. Otherwise, the nuclease might chew away at the plasmid and make screening difficult.
