Please show work!!! You are ready to set up your reaction. You are provided with
ID: 204854 • Letter: P
Question
Please show work!!!
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 2ug DNA in a reaction volume of 50ul. 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/ug). The ApeKI enzyme is supplied at 5,000U/ml.
How many ul of DNA will you add to your reaction tube?
How many ul of 10X buffer will you add to your reaction tube?
How many ul of ApeKI will you add to your reaction tube?
How many ul of dH2O will you add to your reaction tube?
At what temperature will you incubate the reaction?
Explanation / Answer
We will use the formula RT/G to get the required volumes
R = required concentration
T total volume
G given concentration
DNA: 2*50/1000=0.1ul
10 X buffer: 1x*50/10= 5ul
APK1 2ug DNA is used so we will need 5 units of enzyme since for 1 ug 2.5 units is required hence
5*50/5000=0.05ul
you will add the primers and then calculate the total volume added and minus all these volumes from 50 and addd that much water to make it upto 50
temperature will depend on the GC content and the optimal enxyme temp.