Hi Hangameh ,

The whole point of heat inactivation is to enable sequential restriction with two or more enzymes that are not compatible in their restriction conditions (the most common are: temperature, buffer, pH, metal ions and more), without having interference between them, thus avoiding unwanted reactions.

To this end if you restricted your plasmid with PstI, then did heat inactivation as recommended by the manufacturer and then add the second enzyme (naturally in the right conditions), only the second enzyme (I assume that you meant DraI), will perform the wanted restriction digest.

A couple of points worth taking care of when performing this:

1) In some cases the enzymes do not work in the same buffer, and then you need to exchange between the two reactions (the most common is salts and their ionic strength, pH ..). In such a case you should start with a small volume of restriction in the first buffer and first enzyme (e.g - 10ul reaction with the lower ionic strength), followed by increasing the reaction volume and exchanging the buffer to the second one (e.g. - to 25ul while adding the second buffer to 1X). In such a case the best would be to get the suggestion of the double digest tools online from your restriction enzymes vendor (e.g - NEB, Fermentas that have very comprehensive double digest tool https://nebcloner.neb.com/#!/redigest, https://www.thermofisher.com/il/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/double-digest-calculator-thermo-scientific.html

2) Make sure not to exceed a final concentration of 5% glycerol during the second restriction, as the enzymatic activity of some enzymes will be inhibited with high glycerol, and in the worst case may result in Star Activity, hence non specific restriction of the DNA at unwanted sequences.

Best of luck with your restriction,

Ghil

Thank you Ghil this helps a lot.

Dear Hangameh Cassim ,

After the heat inactivation of the 1st enzyme (Pst I) in the reaction mixture , the 2nd enzyme (Dra I) would work definitely provided that the restriction sites for the latter enzyme (Dra I) are present in your DNA sequence and there is no any substance present in the reaction mixture to inhibit/interfere the enzyme.

Warm Regards,

If what you're doing is sequential digestion than I'd recommend skipping the heat inactivation all together and do clean up with spin columns or do ethanol precipitation.

good luck