1. Restriction Enzyme Digestions
Recipes

2. Water (to make final volume)
4 Components
DNA
Enzyme
Buffer
Water (to make final volume)

3. Decide the final volume that you want
(Usually uL)

4. 2. Calculate volume of DNA solution needed
Most digests have ug DNA
1.0 ug = 1000 ng

5. 3. Calculate volume of buffer needed (Buffers usually supplied at 10X
So you need 0.1 x final volume of digest)
BUT BE SURE TO READ THE DIRECTIONS
ON THE TUBE OF BUFFER!!!!!!!

6. 4. Calculate volume of enzyme needed. One unit of enzyme is enough.
(Almost always uL)
BUT BE SURE TO READ THE DIRECTIONS
ON THE TUBE OF ENZYME!!!!!!!

7. 5. Calculate volume of water needed to make final volume.
( = Final Volume – total volume of DNA + Buffer + Enzyme)

8. Decide the final volume that you want
For this example we are choosing 25 uL.
Final Volume = 25 uL

9. 2. Calculate volume of DNA solution needed
What is the concentration of DNA in your sample?
Suppose it is 80 ng/uL.
Suppose you want 500 ng DNA in your reaction.
How many uL is that? = 500 ng/80 ng/uL = 6.25 uL
? = Amount needed/concentration

10. 3. Calculate volume of buffer needed (Buffers usually supplied at 10X
So you need 0.1 x final volume of digest)
BUT BE SURE TO READ THE DIRECTIONS
ON THE TUBE OF BUFFER!!!!!!!
If the buffer is supplied at 10X then 0.1 x 25 uL
= 2.5 uL

11. 4. Calculate volume of enzyme needed. One unit of enzyme is enough.
(Almost always uL)
BUT BE SURE TO READ THE DIRECTIONS
ON THE TUBE OF ENZYME!!!!!!!
Suppose the tube states that 1 uL of enzyme
contains at least 1 unit.
Then use 1 uL of enzyme.

12. 5. Calculate volume of water needed to make final volume.
( = Final Volume – total volume of DNA + Buffer + Enzyme)
Final Volume = 25 uL
DNA volume = 6.25 uL
Buffer volume = 2.5 uL
Enzyme volume = 1 uL
DNA + Buffer + Enzyme =
6.25 uL uL + 1 uL = 9.75 uL
25 uL – 9.75 uL = uL water