You want to cut 1 microgram of a plasmid with 4 units of the restriction enzyme

Question

You want to cut 1 microgram of a plasmid with 4 units of the restriction enzyme HindIII for two hours at each of five different temperatures: 10 degrees celsius, 20 degrees celsius, 30 degrees celsius, 40 degrees celsius, and 50 degrees celsius. The plasmid stock is 250 nanograms/microliter, the enzyme is 10 units/microliter, the reaction buffer is 5x, and the total volume of each reaction is 20 microliter. Using the New Englands Biolabs catalog, indicate your plan for setting up these reactions.

Explanation / Answer

Concentration of DNA is 250ng/ µl and we need to add 1µg DNA so the volume of DNA we will use is 4µl

For 20µl reaction, we need to use 4µl of 5X buffer

Given the concentration of enzyme is 10U/µl. we need to use 4U of HindIII for a reaction so for each reaction we will use 0.8 µl of the enzyme.

After setting the reaction, keep the reaction tubes at the desired temperature and after 2 hours observe the digestion on the agarose gel

DNA 4µl HindIII enzyme 0.8µl 5X buffer 4µl Autoclaves H2O to make volume 20µl 11.2µl Total volume 20µl

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