Quick Protocol for Monarch® Plasmid Miniprep Kit (NEB #T1010)
Pellet 1–5 ml bacterial culture by centrifugation at 16,000 x g for 30 seconds.
Discard supernatant.
Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1).
Vortex or pipet to ensure cells are completely resuspended.
There should be no visible clumps.
Add 200 μl Plasmid Lysis Buffer (B2), gently invert tube 5–6 times, and incubate at room temperature for 1 minute.
Color should change to dark pink, and solution will become transparent and viscous.
Do not vortex.
Add 400 μl of Plasmid Neutralization Buffer (B3), gently invert tube until neutralized, and incubate at room temperature for 2 minutes.
Sample is neutralized when color is uniformly yellow and precipitate forms.
Do not vortex.
Centrifuge lysate at 16,000 x g for 2–5 minutes.
For culture volumes >1 ml, we recommend a 5 minute spin to ensure efficient RNA removal by RNase A.
Pellet should be compact; spin longer if needed.
Carefully transfer supernatant to the spin column and centrifuge at 16,000 x g for 1 minute.
Discard flow-through.
Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1.
Centrifuge for 1 minute at 16,000 x g. Discarding the flow-through is optional.
Add 400 μl of Plasmid Wash Buffer 2 and centrifuge at 16,000 x g for 1 minute.
Transfer column to a clean 1.5 ml microfuge tube.
Use care to ensure that the tip of the column does not come into contact with the flow-through.
If there is any doubt, re-spin the column for 1 minute.
Add ≥ 30 μl DNA Elution Buffer to the center of the matrix.
Wait for 1 minute, then spin for 1 minute at 16,000 x g to elute the DNA.
Nuclease-free water (pH 7–8.5) can also be used to elute the DNA.
