Restriction Enzyme Digestion of Plasmid DNA
Reagents Required
-
Purified plasmid DNA
-
Restriction enzyme(s)
o Choose enzymes that
produce clear, distinguishable band patterns on agarose gel.
o Use enzymes with unique
cut sites to confirm plasmid structure or insert presence.
o Ensure buffer and
temperature compatibility, especially for double digests.
-
10× or 5× reaction buffer
(provided with enzyme)
-
Nuclease-free water
-
BSA (if required by enzyme)
Equipment
-
Microcentrifuge tubes (1.5 mL)
-
Pipettes and sterile filter
tips
-
Thermal cycler or Water Bath
-
Ice bucket
Steps:
1. Standard
Reaction Setup (20 µL Total Volume)
|
- |
Plasmid DNA |
0.5–1 µg (~1–5 µL) |
|
- |
10 × Restriction Buffer |
2 µL |
|
- |
Restriction Enzyme |
0.5–1 µL |
|
- |
Nuclease-free Water |
Up to 20 µL total |
2. Reaction
Assembly
-
Set up the reaction on ice in a
sterile 1.5 mL microcentrifuge tube.
-
Add in the order of water →
buffer → DNA → enzyme gently by tapping or flicking (do not vortex enzymes).
-
Gently mix by pipetting or
flicking.
3. Incubation
-
Incubate at enzyme-specific optimal
temperature for 1 hour if restriction digestion is for electrophoresis.
Storage
-
Store digested DNA at –20°C.
-
Store enzymes and buffers as
per manufacturer’s instructions (typically –20°C).