Monarch Plasmid Miniprep Kit protocol

Learn how to do a plasmid miniprep using the Monarch Plasmid Miniprep Kit.

Script

Ana Egana, PhD:

My name is Ana Egana.  I am the technical support manager at New England Biolabs.  I am also the production manager for the nucleic purification Monarch product line.  Today in this short video we are going to show you the basic steps that you need to follow to do a plasmid purification using our Monarch Plasmid Miniprep Kit. 

You will need to add 4 volumes of ethanol to one volume of Plasmid Wash Buffer 2 according to the instructions on the bottle’s label.

Now to the first step, the pelleting.  You will need to pellet between 1 and 5 mls of bacterial culture by centrifugation at 16,000 x g (~13,000 RPM) for 30 seconds. After that make sure that you carefully discard the supernatant.

The next step will be the resuspension step.  Resuspend the bacterial pellet in 200 μl of Plasmid Resuspension Buffer [B1]. You can vortex the tube or pipette it up and down to ensure the cells are completely resuspended. It is important to resuspend the cells completely in order to achieve complete lysis in the following step.

To lyse the cells you will add 200 μl of Plasmid Lysis Buffer [B2] to the cell suspension.  Immediately and gently invert the tube 5-6 times until the color changes to dark pink and the solution is viscous. Please do not vortex the solution since this can shear the DNA!  Now you will need to incubate for 1 minute at room temperature to ensure complete lysis.  Move quickly to the next step to avoid denaturing the plasmid.

To neutralize the lysate add 400 μl of Plasmid Neutralization Buffer [B3], and again gently invert the tube until the color is uniformly yellow and a precipitate forms. It is very important that you mix the solution until the color is uniformly yellow. Please do not vortex the solution! Now you will need to incubate for 2 minutes at room temperature, then centrifuge for 2-5 minutes.

Now to the binding step.  You will carefully transfer the supernatant, which contains the plasmid DNA, to a spin column, and centrifuge for 1 minute. The DNA is now bound to the column's matrix. You will remove the column, and you will discard the flow-through.

Now the washing begins. Re-insert the column into the collection tube, and add 200 μl of Plasmid Wash Buffer 1.  Centrifuge for 1 minute. You may discard the flow-through at this point, but it is optional. Add 400 μl of Plasmid Wash Buffer 2, and centrifuge for 1 minute.  You will transfer the column to a clean microcentrifuge tube, making sure that the tip of the column does not come into contact with the flow-through.

Now for the elution step.  You will add 30 or more μl of DNA Elution Buffer to the center of the column membrane. Incubate for the solution for 1 minute at room temperature. This incubation step is important to guarantee maximum yields. Centrifuge for 1 minute and collect the flow-through.  The flow-through now contains your purified plasmid DNA. 

If you need additional information on this protocol, you can check the manual on the product webpage.  If you have any difficulties you can always contact NEB Technical Support. 

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