It is often recommended to heat inactivate restriction enzymes, ligase, phosphatase at 65 degree for 10 mins or so and use the DNA for subsequent steps of cloning. How efficient is the heat inactivation? If any protein is heat inactivated, can we skip phenol-chloroform extracion of proteins from the reaction mix? Does it have a risk of residual restriction enzyme or say CIP which might interfere? When the reaction mix is stored at a cold temperature, is there is any chances of renaturation of the inactivated proteins?
The heat inactivation works well enough to use the DNA in downstream steps such as electroporation or heat shock transfection. I have never had a problem getting my cells to take the plasmid without doing Phenol chloroform. If you are storing the DNA at between 4 and -20 there shouldn't be any issue with renaturation.
These enzymes are usually well-characterized so I suppose heat inactivation is sufficient to get inactivate the enzymes before proceeding with subsequent cloning steps. But don't forget that buffer components from earlier reactions might also interfere in later cloning steps. In case that buffers in two subsequent steps are incompatible, I would clean up my DNA with phenol/chloroform extraction in between.
hi
while cloning if the buffers are not compatible you have to go through a phenol chloroform extraction step or else its not required!!
CIP needs to be deactivated by heat. if it remains no cloning is gonna be sucessful!!
after all enzymatic reactions u can run the products and gel elute
however in case you are not gel eluting with a kit you have to do phenol chloroform to remove the enzymes, buffers and the small cut fragments... nything other than ur desired fragments for cloning...
Hi Tias,
It depends on the enzymes: look at provider website or catalog. For instance in the Biolabs catalog, they recommend 20 min at either 65°C or 80°C for restriction enzymes, and in some cases, they indicate that the enzyme cannot be heat inactivated. Of course if you purify your fragments on a gel you don't need to inactivate the restriction enzymes. In my hands, heat inactivating a ligation is counterproductive: it reduces the number of transformants, probably because at some point during the heating, DNases get activated and degrade the DNA (whose concentration in ligation reactions is generally low), while the presence of non heat-inactivated ligase during the transformation does not appear to be a problem.
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