1. You are ready to set up your reaction. You are provided with a stock of plasm
ID: 208482 • Letter: 1
Question
1. 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 (dH2O) to use, as needed.
As noted above, you want to cut 2mg DNA in a reaction volume of 50ml. 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/mg). The ApeKI enzyme is supplied at 5,000U/ml.
a. How many ml of DNA will you add to your reaction tube?
b. How many ml of 10X buffer will you add to your reaction tube?
c. How many ml of ApeKI will you add to your reaction tube?
d. How many ml of dH2O will you add to your reaction tube?
e. At what temperature will you incubate the reaction?
Explanation / Answer
a) Since 2mg of DNA is required, and 1mL has 1mg of DNA, 2 mL of stock of plasmid
b) since the final reaction volume is 50 mL and the stock is 10X, to make it into 1X use the formula C1V1=C2V2, where C1=10X, V1=?, C2=1x and V2=50. This implies that the amount of buffer to be added in the reaction tube = V1=5mL.
c) For 1mg DNA, it is given that you require 2.5U of enzyme. Therefore for 2mg of DNA you require 2.5Ux2=5 units. Now, the enzyme is supplied at 5000 units per ml. So for 5 units you will need 5/5000 = 0.001mL of enzyme.
d) The remaining volume has to be made with dH2O. The required amount is 42.999ml
e) 75C. At this temperature the enzyme shows optimal activity
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