You are ready to set up your reaction. You are provided with a stock of plasmid

Question

You are ready to set up your reaction. You are provided with a stock of plasmid DNA that is at a concentration of 1mg/ml. You have stock enzyme buffer that is 10X (meaning it is ten-times more concentrated than you need it to be in the actual reaction). You have deionized water (dH_2 O) to use, as needed. As noted above, you want to cut 2 mu g DNA in a reaction volume of 50 mu l. To make sure you get complete cutting, you'll use an excess of enzyme so that you use 2.5 units per microgram of DNA (2.5U/mu g). The ApeKI enzyme is supplied at 5,000 U/ml. How many mu l of DNA will you add to your reaction tube? How many mu l of 10X buffer will you add to your reaction tube? How many mu l of ApeKI will you add to your reaction tube? How many mu l of dH_2 O will you add to your reaction tube? At what temperature will you incubate the reaction?

Explanation / Answer

2 uL of DNA is needed.

5 uL of 10X buffer should add

0.4 uL of ApeKI should add

40.6 uL of dH2O is needed

750C is the optimal temparature for ApeKI restriction enzyme.

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