Question

You are adding 2 μL of a 10X restriction buffer to a reaction that will total 20 μL. What is the final concentration of buffer in the reaction (in terms of “X”)?

Add 4 μL of 0.1 ug/uL DNA to each reaction tube. How many ug of DNA are being digested in this reaction?

When you have set up all of your reactions, bring your reactions in your ice bucket to the front of the room to add 1 μL of EcoRI (15 units/uL), BamHI (40 units/uL), and HindIII (50 units/uL) to the appropriate tubes. How many units of each enzyme are in each reaction?

Enzymes are stored in special containers that maintain the -20°C freezer temperatures when taken out of the freezer. Enzyme storage buffers contain 50% glycerol as an antifreeze. Enzymes must be stored properly and treated carefully so that they do not denature and lose activity. However, if there is too much glycerol in the final reaction ( >10%), it may cause the enzyme to cut the DNA nonspecifically (in places other than the recognition sequence for the enzyme). This is called “star” activity. Give that you are adding 1 L of enzyme that is stored in 50% glycerol to a 20 uL reaction, calculate the final percentage of glycerol in the reaction. Should you be worried about star activity?

Answer 1

In this question the total volume of the reaction is 20l

Then

A:Restriction buffer concentration =10X

Addition of 2l to the reaction

Then According to dilution rxn

I.e M1XV1= M2XV2

M=concentration and V= volume of the solution

10 X 2 = M2 X 20

M2 = 1

Therefore final concentration is 1X

B: Concentration of the DNA is given as .1ug/l using of 4l of this solution gives the .4ug DNA in the reaction

C EcoRI (15 units/uL), BamHI (40 units/uL), and HindIII (50 units/uL) of concentration and we were using 1l of each enzyme.So unit of these enzymes are as follows 15unit of EcoR1,40unit of BamH1,50unit of Hind111 in the reaction.

D: enzyme stock contain 50% glycerol

Then M1 X V1 = M2 X V2

50 X 1 = M2 X 20

M2 = 2.5

Therefore final glycerol concentration in the solution is 2.5%