BIOLOGY 40-PROTOCOL #7-Revised
Summer, 2007
Name:________________________________________
Date:_________________________
Protocol 7 - Restriction Enzyme Digest of DNA
Digest
ddH2O 10 X Buffer
DNA
Enzyme
FINAL
VOLUME
1. Eco RI
Type:
2. Pst I
Type:
Type:
Supplies needed:
Equipment needed:
- restriction enzyme buffers (10X)
- microcentrifuge
- restriction enzymes
- P-10, P-100 micropipettors
- DNA to be cut by restriction enzyme
- water bath at optimal temperature
- 1.5 ml sterile microcentrifuge tubes
- rack to hold microfuge tubes
- ddH2O
Important Notes to Remember:
 Use gloves and sterile technique throughout the protocol to avoid contamination.
 Carry the enzyme from the freezer on ice. Only remove the enzyme from the freezer
or from the ice when you are prepared to add it. When enzymes are off the ice for a
prolonged period, they can lose activity.
 Use a different pipette tip for each component of the reaction so as to NOT
contaminate the stock solutions.
 If you are using genomic DNA, avoid excessive pipetting and spinning in order to
avoid sheering which may cause undesired breakage.
 Each restriction enzyme works optimally with a certain buffer (pH, salt concentration,
ions, etc.) and at a given temperature (most, but not all, work best at 37oC). See chart.
1. Determine the final volume of the reaction you wish to perform (e.g. 50 μl). Then, based
on the stock concentrations, calculate the necessary amount of each component of the
reaction, calculating the amount of water needed last.
2. Add the components to your 1.5 ml microcentrifuge reaction tube in the following order:
 ddH2O
 Buffer
 DNA
3. Mix these components by gently pipetting the sample up and down.
4. Add the enzyme to the digestion mix and incubate the reaction at the appropriate
temperature (e.g. 37oC) for the appropriate amount of time (no more than 2 hours).
5. After the digestion time has elapsed, stop the reaction by placing the sample at -20°C for
30 minutes or overnight.
6. A portion of the reaction mixture can be checked for cutting by loading and running on an
agarose gel.