Aug 12, 2022

DNA Purification (NEB)

This protocol is a draft, published without a DOI.
  • 1University of Wisconsin - Stout
  • Yeast ORFans CURE
Brian Teague
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Protocol Citation: Brian Teague 2022. DNA Purification (NEB). protocols.io https://dx.doi.org/
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 11, 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 Monarch PCR and DNA Cleanup Kit - 50 prepsNew England BiolabsCatalog #T1030S

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!
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 200 µL of sample, mix it with 1000 µL of binding buffer.


Insert the column into the collection tube and load the sample onto the column.
Centrifuge 16000 x g, 00:01:00 . Discard the flow-through in the chemical waste container.

1m
Re-insert the column into the collection tube. Add 200 µL of Wash Buffer.

Centrifuge 16000 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 10 µL of Elution Buffer to the center of the silica matrix. Wait 00:01:00 , then centrifuge 16000 x g, 00:01:00

2m
Quantify your purified DNA on the Nanodrop.