In every leaflet of restriction enzyme we find a booklet about the reaction composition, incubation temperature and reaction guidelines for every specific restriction enzyme. Here, I have question, like for some of restriction, it is written that “Inactivation temperate is 80C for 20 minutes”.
So my question is that,
1. Will the restriction enzyme will be destroy completely or inactivate (Restriction enzyme will not be active at 80C temper but can be re-activate with another incubation at its own incubation temperature) but will be at 80 for 20C ?
Dear Nahid,
I think you wanted to write 80°C for 20 minutes.
Inactivation Via heat treatment:
Certain enzymes can be inactivated by heating. …
Inactivation by heating depends on the enzyme you use!
These are not always the same inactivating conditions!
There some other Enzyme inactivating methods!
Try EDTA inactvating!
Best regards,
Thomas
Dear Nahid
“Thermal Inactivation MnlI is inactivated by incubation at 65°C for 20 min.”
Under this conditions the enzyme will be inactivated or denaturated.
After this heat inactivation this enzyme will not work again, it is denaturated!
Best regards,
Thomas
Dear Sir,
Yes, but i have a confusion about the term inactivation. So, if i run a mixture in inactivation temperature and i didn't get any restriction digestion as because the enzyme was inactivated so Can i get any digestion from that mix if i run it again at specific incubation temperature ?
Your Obedient,
nahid
Dear Nahid,
As I wrote to you:
After heat inactivation (= denaturation) the enzyme can not work again, because the enzyme is denaturated!
You could us the same PCR product sample (after PCR and unsuccessful digestion) again. In this case you must give new well working Enzyme to this sample!
After this, you must incubate the same sample under the right digestion conditions.
Best regards,
Thomas
Hi there,
Enzyme heat inactivation step leads to an irreversible effect. The goal being to both inactivate enzyme activity and also to prevent any binding of the enzyme to DNA which might interfere with downstream applications.
Dear Sir Thomas M.C. Binder,
Thank you Sir for your comment and may be i need to prepare new digestion again with that enzyme and pcr product.
Your Obedient,
nahid Islam
Dear Sir Dominique Liger,
Thank you Sir for the comment the mechanism. Now its clear to me.
Sincerely,
nahid Islam
Hi all!
Connecting to this topic I have a question.
Generally 65°C for 20 mins inactivates the majority of restriction endonucleases, however some requires 80°C for 20 mins. I suppose during a double digestion, enzymes with different inactivation temperature (65 and 80°C) can be both inactivated at 80°C. There are restriction endonucleases which cannot be heat-inactivated. In that case EDTA (pH 8.0) in 20mM final concentration is an option. What if during my double digestion one of my enzymes can be heat-inactivated and the other one requires EDTA? Just add EDTA (pH 8.0) in 20mM final conc.? It will inactivate both?
Thank You in advance!
Dear Mark,
In a double digestion with these both inactivation temperatures, 80°C for 20 minutes will inactivate both endonucleases.
In the second case, I would try to denaturate with EDTA (pH 8.0) in 20 mM final concentration in addition with 65°C for 20 minutes (or 80°C for 20 minutes).
Best regards,
Thomas
To whom it may concern with the answer of the question;
Thermal inactivation is an optional choice. In my opinion, you should inactivate your digested products so as to use them in downstream analyses. For example you may use pcr products for digestion and because of the amount of DNA in the PCR product is not known exactly, practically you may want to add some DNA into tube for better resolution on the gel, so the thermal inactivation is not neccessary in this step.
Hi
In my work I have EcoRI and Hind III , these two enzymes with different inactivation temperature (65 and 80°C) .
I depend on 80°C to inactivate these enzymes, and every things OK.
If any body can support me with articles as references to my thesis.
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