Biology Forum › Molecular Biology › Inactivate an enzyme
- AuthorPosts
- April 11, 2012 at 1:53 am #16342
foufouletteParticipantHi 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 ?
- April 11, 2012 at 9:18 am #110585JackBeanParticipant
What do you think? Will the phosphatase do something if you have there plenty of ATP and not-dephosphorylased insert DNA?
- April 11, 2012 at 12:35 pm #110587foufouletteParticipant
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.
- April 11, 2012 at 1:14 pm #110588JackBeanParticipant
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.
- April 12, 2012 at 12:01 am #110595foufouletteParticipant
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 !
- April 12, 2012 at 8:39 am #110599JackBeanParticipant
You must inactivate it before ligation. After ligation is the phosphorylase harmless. 🙄
Expect low yield, if you’ll get something at all.
- April 13, 2012 at 2:01 am #110606foufouletteParticipant
I made transformation and i had colonies but what do you mean by yield ?
- April 13, 2012 at 8:06 am #110610JackBeanParticipant
The number of ligated vectors should be lower than if you inactivated the phosphatase. Let us know, if you’ve got something positive.
- AuthorPosts
You must be logged in to reply to this topic.