Monarch® PCR & DNA Cleanup Kit Protocol

Learn how to isolate DNA from your enzymatic reactions, including PCR, using the Monarch PCR & DNA Cleanup Kit (5 µg).

Script

Nathan Tanner, PhD:

My name is Nathan and I am a research scientist here at NEB and today I'm going to show you how to use our Monarch PCR & DNA Cleanup Kit.  Before you get started, add 4 volumes of ethanol to one volume of DNA Wash Buffer according to the instructions on the bottle's label. All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM)

First, we'll dilute the sample. We recommend starting with a DNA sample size of 20-100 μl. Add the recommended amount of DNA Cleanup Binding Buffer to your sample. The ratio of DNA Cleanup Binding Buffer volume to add into your sample volume is dependent on the size and type of DNA in your sample.

Refer to the table on the protocol card to determine this amount. If you are working with DNA sample volumes smaller than 20 μl, you will need to first increase these volumes to at least 20 μl by adding TE buffer. If your sample is greater that 800 μl after being diluted with binding buffer, you will need to load the column with a portion of it, spin, and then load the rest, as the column reservoir may not be able to contain all of it at once.  Mix well by pipetting up and down or flicking the tube.

Now we will bind the DNA to the column.  Load the sample onto a column seated in a collection tube, and close the cap. Spin for 1 minute. The DNA is now bound to the column's matrix. Remove the column from the collection tube, and discard the flow-through.

Now we'll wash the DNA.  Re-insert the column into a collection tube, and add 200 μl of DNA Wash Buffer. Spin for 1 minute. You can discard the flow-through at this point, although it is optional. Repeat the wash step by adding 200 μl of DNA Wash Buffer, and spinning again for 1 minute.

And now we will elute the DNA from the column.  Transfer the column to a clean microfuge tube, making sure that the tip of the column does not come into contact with the flow-through, in order to avoid contamination. To elute the DNA from the column's matrix, add 6 or more μl of DNA Elution Buffer to the center of the column matrix, and incubate for 1 minute. This incubation step is important for maximizing yields. Spin for 1 minute and collect flow-through, which contains your purified DNA.

For more information, check out the product manual on our website or contact NEB for technical support. 

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