Aug 12, 2022

Public workspaceDNA Purification (NEB)

This protocol is a draft, published without a DOI.
  • Brian Teague1
  • 1University of Wisconsin - Stout
  • Yeast ORFans CURE
Brian Teague
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Protocol Citation: Brian Teague 2022. DNA Purification (NEB). protocols.io https://protocols.io/view/dna-purification-neb-ce6sthee
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 12, 2022
Last Modified: August 12, 2022
Protocol Integer ID: 68530
Keywords: pcr, purification, monarch, spin column, dna purification, restriction enzyme, dna, dna from an enzymatic reaction, neb, enzyme, miniprep, enzymatic reaction
Abstract
This protocol purifies DNA from an enzymatic reaction (like a PCR or a digestion with a restriction enzyme.) It is NOT an miniprep -- if you are trying to purify DNA from a culture of E. coli, you are in the wrong place!
Materials
  • Binding buffer
  • Wash buffer
  • Elution buffer
  • One spin column and collection tube per cleanup
  • Chemical waste container (50 ml conical)

The buffers and spin column are from the ReagentMonarch PCR and DNA Cleanup Kit - 50 prepsNew England BiolabsCatalog #T1030S

Troubleshooting
Safety warnings
The binding buffer may cause irritation to skin and eyes. Additionally, we are shedding nucleases -- enzymes that degrade DNA -- all the time. Wear lab coats, gloves and safety glasses.

The flow-through (containing the binding buffer or wash buffer) cannot go down the drain. Dispose of it as chemical waste, per the directions of your instructor.
Make sure you are using the "Monarch PCR & DNA Cleanup Kit", not the Monarch Plasmid Miniprep Kit. They come in identical boxes -- read the label!
Critical
Mix the ENTIRE DNA sample with 5 times its volume of Binding Buffer. Pipette up and down several times to mix the sample thoroughly.

Note
Ie, if you had Amount200 µL of sample, mix it with Amount1000 µL of binding buffer.


Insert the column into the collection tube and load the sample onto the column.
Centrifuge Centrifigation16000 x g, 00:01:00 . Discard the flow-through in the chemical waste container.

1m
Re-insert the column into the collection tube. Add Amount200 µL of Wash Buffer.

Centrifuge Centrifigation16000 x g, 00:01:00 . Discard the flow-through in the chemical waste container.

1m
Repeat steps 5 and 6 once.
Transfer the column to a clean 1.7 ml microcentrifuge tube.
Add Amount10 µL of Elution Buffer to the center of the silica matrix. Wait Duration00:01:00 , then centrifuge Centrifigation16000 x g, 00:01:00

2m
Quantify your purified DNA on the Nanodrop.