1. Transfer the following solutions in a micro centrifuge tube.
2. Incubate the mixture at 37 o C for 1 h to overnight. Keep the tubes in -4o C freezer or in -20o C freezer, after the incubation.
Make sure that the restriction enzyme does not exceed more than 10% of the total reaction volume, Otherwise the glycerol and the EDTA in the enzyme storage buffer may inhibit digestion process.
Differences Encountered in Real Laboratory
After performing the experiment, confirm the Digestion of DNA by running a small amount of it in agarose gel with an undigested standard DNA.
Some restriction enzymes require BSA. In such cases make sure that, it is added to the reaction mixture. Restriction enzymes that do not require BSA for optimal activity are not adversely affected if BSA is present in the reaction.
Before performing the experiment, check whether the restriction enzymes have star activity or not.
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