Inactivate an enzyme

[foufoulette]

Hi everyone !

Please, i need your opignon about what i've done. Is it correct to don't inactivate enzyme (phosphatase) after a diphosphatasing of a plasmid and do the next step which is ligation, without inactivation ? is this could affect my results ?

[JackBean]

What do you think? Will the phosphatase do something if you have there plenty of ATP and not-dephosphorylased insert DNA?

[foufoulette]

The enzyme could do something if the DNA insert is not dephosphorylased, so what shall i do ? cause i don't need to dephosphorylate the insert.

[JackBean]

You do not only don't need but must not dephosphorylate your insert, because then the DNA ligase won't be able to ligate the fragments together. It needs the phosphate on the DNA strand.

[foufoulette]

Ok i understand but actually I had inactivated phosphatase in 65°C after ligation and it's correct i think, now i'm waiting for the results !

[JackBean]

You must inactivate it before ligation. After ligation is the phosphorylase harmless.

Expect low yield, if you'll get something at all.

[foufoulette]

I made transformation and i had colonies but what do you mean by yield ?

[JackBean]

The number of ligated vectors should be lower than if you inactivated the phosphatase. Let us know, if you've got something positive.