Joyce: T-tailing pBSKS: Difference between revisions
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(New page: Procedure #Obtain about 20 ug of pBSKS #Cut with a blunt cutting restriction enzyme (like EcoRV) #Extract the DNA with 1X phenol and 2X ether #Precipitate the DNA) |
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[[Procedure]] | [[Procedure]] | ||
#Obtain about | #Obtain about 10 ug of pBSKS | ||
#Cut with a blunt cutting restriction enzyme (like EcoRV) | #Cut with a blunt cutting restriction enzyme (like EcoRV) | ||
# | #Precipitate the DNA using NaOAc and ethanol | ||
#Precipitate the DNA | #T-tail the DNA using: | ||
**10 ug of pBSKS | |||
**10 ul of 10X Taq buffer (no MgCl<sub>2</sub>) | |||
**0.5 ul 100 mM dTTP | |||
**6 ul 25 mM MgCl<sub>2</sub> | |||
**1 ul 5 U/ul Taq | |||
**Sterile dH<sub>2</sub>O to 100 ul | |||
#Incubate at 72°C for 2 hours | |||
#Extract with 1X phenol and 2X ether | |||
#Precipitate the DNA with NaOAc and ethanol | |||
#Resuspend in 150 ul of sterile dH<sub>2</sub>O | |||
#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. | |||
Revision as of 15:04, 10 September 2009
- Obtain about 10 ug of pBSKS
- Cut with a blunt cutting restriction enzyme (like EcoRV)
- Precipitate the DNA using NaOAc and ethanol
- 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
- Incubate at 72°C for 2 hours
- Extract with 1X phenol and 2X ether
- Precipitate the DNA with NaOAc and ethanol
- Resuspend in 150 ul of sterile dH2O
- 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.
