Hello !
I have linearized a plasmid overnight using a single-cutting restriction enzyme (SalI). I then inactivated the enzyme at 70C for 20 minutes, and stored the linearized plasmid at -20C.
I am planning on using it several times for transformation experiments in Neisseria meningitidis, which is naturally competent. I know that using the freshly linearized plasmid gives good transformation efficiency. However, I am worried about the plasmid closing itself after some time, since SalI generates complementary ends.
Has anyone ever tested this ? My sample is precious and I can't really afford to do an electrophoresis to check for the plasmid conformation everytime I need to use it.
Thanks for the help
Hi there,
If you are sure of the completion of the initial digest there is no problem. Uncatalysed self ligation is just impossible (Ligase uses coupling with ATP hydrolysis energy to make it possible).
Hello Martin,
I agree with Dr.Dominique, Two ends of completely Digested and Purified linear Plasmid can not ligated again anyway.
All the best,
Hamed
Well, Dominique ir right, that it will not ligate back, but the sticky ends formed by SalI may bind back together. There's a simple solution though - just heat the DNA to 65°C for 5-10 min and then cool it down quickly on ice.
I do this even before ligation to make sure that the ends are free and available for binding with other molecules.
Hi
yes this self ligation is impossible.
I had digested vector that use it several moons later. even ligase hardy can do that!!
Hey Martin!!
You can try by adding Phosphatase to your linearized plasmid after the restriction enzyme inactivation step. You can use SAP or CIP. Dephosphorylation prevents religation of linearized plasmid DNA.
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