4 replies to this topic

[planktonica]

The protocol in my lab does not state any inactivation at 65°C for 10 mins. However, I found in some general resources online that AP must be heat inactivated. Then I asked around to some other labs and they don't inactivate.

Is this step critical?

I had successful cloning before, but this time I am having a hard time and I am revising every single step and protocol and this AP inactivation popped out as some potential culprit.

What do you think?

[perneseblue]

planktonica, on Mar 5 2009, 09:24 AM, said:

The protocol in my lab does not state any inactivation at 65°C for 10 mins. However, I found in some general resources online that AP must be heat inactivated. Then I asked around to some other labs and they don't inactivate.

Is this step critical?

I had successful cloning before, but this time I am having a hard time and I am revising every single step and protocol and this AP inactivation popped out as some potential culprit.

What do you think?

Yes, you should inactivate the phosphotase. If left active, it can dephosphorylate all your DNA.
However, not all phosphotase can be heat inactivated. CIP is heat resistant and has to be removed by phenol-chloroform or column purification.

[T C]

Hey

After CIP treatment, I either run the vector on the gel or column purify immediately after treatment so don't need to inactivate in this case.

Best
TC

[planktonica]

I usually do column spin purification. But this time I did the heat inactivation AND purification step. I decided not to run a gel because I didn't want to get depressed! So I just continued with transformation. Hopefully get something tomorrow.

Thanks!

[scolix]

It is better to inactivate all enzymes after a reaction.