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Key Concepts
A restriction enzyme digest will be used to cut the DNA of the pET28b vector, creating ‘sticky ends’ at a specific site in the plasmid. We will use the restriction enzyme BamHI to cut pET28b at a site where our GFP PCR product can be inserted later using the Gibson Assembly cloning method.
Materials
- pET28b plasmid – aliquots @ -20 °C
- Restriction enzyme: BamHI from NEB – aliquots @ -20 °C
- 10X NEB CutSmart Buffer – aliquots @ -20 °C
- Nuclease-free water
Procedure
Digestion Reaction Mixture
|
|
Stock concentration | Final reaction concentration |
Amount to use (uL) |
|
NEB Cutsmart Buffer |
10X | 1X | x |
| 250 ng/uL | 1 ug |
y |
|
|
BamHI enzyme |
20 U/uL | 20 U |
z |
|
Nuclease-free water |
– | – |
50-x–y–z |
|
Total Reaction Volume |
50 µL |
Important note: Enzyme stocks (e.g. BamHI) are extremely temperature-sensitive! They should be kept on ice at ALL times when not in use. Also, the enzyme is suspended in a high-percentage glycerol stock, which tends to stick to the sides of your tip. To minimize errors in pipetting these glycerol stocks, wipe the side of the tip off within the enzyme tube and only dip the pipette tip just below the surface.
The NEB CutSmart Buffer is stored in the freezer. Completely thaw the buffer and vortex before pipetting to ensure that it is well-mixed (freezing creates concentration gradients).
- Make the digestion reaction mix in a 1.5mL tube. Use deionized water to bring volume up to 50µL. *Add the BamHI enzyme last!
- Mix by gently pipetting up and down a few times.
- Incubate at 37°C for ~1 hour.
- Perform a PCR cleanup on your digested DNA (see next protocol for more information).
- Save the digested DNA at 4°C for use in the Gibson Assembly reaction.