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 .
