Team:LMU-Munich/Notebook/Protocols/5 Restriction digest




Restriction enzyme digestions were assembled as follows:

plasmid DNA 1µg

restriction enzyme 1µl

10X buffer 2µl

BSA (10µg/µl) 0.2µl

nuclease-free water to final volume 20µl

Table 1. Restrictionenzymes with data about their activity in selected buffers. Optimal also taken from following webpage especially if double digestion wanted:

Enzyme Activity in buffer A Activity in buffer B Activity in buffer C Activity in buffer D Activity in buffer H Activity in buffer MC
XbaI 50-75 75-100 75-100 100 - 100
EcoRI 25-50 50-75 50-75 50-75 100 100
PstI 10-25 50-75 50-75 50-75 100 25-50
SpeI 75-100 100 75-100 75-100 - 100


Vortex all reagents before use. (especially the enzymes)

1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube

2. Add restriction enzyme buffer.

3. Add BSA.

4. Add DNA.

5. Add each enzyme.

Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.

6. Incubate for 2 hours at 37°C.

7. Incubate for 20 mins at 80°C to heat inactivate enzyme. This step is sufficient to inactivate even Pst I.

8. Incubate 4°C until you pull the reaction out of the thermal cycler. (Thermal cycler can be used for a installed program for digestion, even overnight)