Sep 22, 2020
Agarose Gel DNA Extraction (QIAquick)
This protocol is a draft, published without a DOI.
- 1University of Arizona
- Yoon Lab
Protocol Citation: Kenneth Schackart 2020. Agarose Gel DNA Extraction (QIAquick). protocols.io https://protocols.io/view/agarose-gel-dna-extraction-qiaquick-bc9yiz7w
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: In development
We are still developing and optimizing this protocol
Created: March 05, 2020
Last Modified: September 22, 2020
Protocol Integer ID: 33816
Keywords: gel extraction, Qiagen, QIAquick, DNA extraction, DNA purification,
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Abstract
Extract DNA from agarose gel using Qiagen® QIAquick® Gel Extraction Kit. The kit can be used for centrifuge processing or vacuum processing. The steps given here are only for centrifuge processing.
Link to the original product website is provided in the Exernal Link field. PDF file of full protocol from manufacturer is attached (available online). I do not claim any credit for the development of the kit or protocol. Please refer to the original protocol provided by Qiagen for any clarifications.
Attachments
Image Attribution
Karolina Fok - Own work, CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=84931094
Guidelines
This protocol is for the purification of up to 10 µg DNA of length 70 bp - 10 kbp.
Add 96-100% ethanol to buffer PE before use.
All centrifugation steps are carried out at 17900 x g or the highest setting if your centrifuge does not support this speed.
Materials
MATERIALS
Qiagen QIAquick Gel Extraction Kit
- 95 % volume -100 % volume Isopropanol (IPA).
- 1.5 mL centrifuge tube(s)
- Nuclease free water
- Ethanol for suspending Buffer PE before use
Safety warnings
- Gloves must be worn at all times.
- Wear proper PPE when excising gel band if using a UV light source for fluorescence. (In our lab we wear a lab coat, gloves, and polycarbonate full-face mask).
- Acute signs of UV exposure damage include redness and inflammation (like a sunburn), take note of this and address any deficiency in PPE.
- Long-term damage due to UV exposure includes increased chance of skin cancer and eyesight damage.
Before start
- Run gel electrophoresis to generate DNA band(s).
- Set heat block to 50 °C .
- Obtain 95 % volume -100 % volume Isopropanol (IPA).
Excise Gel
Excise Gel
Label and weigh 1 uncolored 1.5 mL centrifuge tube for each gel band to be exracted.
Using a clean razor blade, excise DNA band from gel and place in corresponding 1.5 mL centrifuge tube. Try to cut the gel as close to the DNA band as possible
Safety information
It is critical to avoid exposure of skin or eyes to UV irradiation. When using UV light source for fluorescence ensure that all skin is covered. Wear a full-face mask made of a UV blocking material such as polycarbonate. Be aware of others around you to avoid accidentally exposing them to UV irradiation.
Note
It may be best to use a dulled razor blade when cutting to avoid damage to transilluminator surface.
Weigh the 1.5 mL tube with the DNA gel slice, and determine weight of gel.
Note
Subtract the tube weight found in step 1 to detemine gel weight.
Add 3 volumes Buffer QG to 1 volume gel. For >2% agarose gels, add 6 volumes Buffer QG instead of 3.
Note
100 mg gel ~100 µL
Dissolve Gel
Dissolve Gel
Incubate at 50 °C for 00:10:00 or until gel has completely dissolved.
Vortex every 2-3 min to help dissolve gel.
After gel slice has dissolved, check the color.
- If color is yellow, move to next step.
- If color is orange or violet: add 10 µL 3 Molarity (M) sodium acetate , 5 , and mix.
Add 1 volume isopropanol to the tube and vortex mix.
Bind and Spin DNA
Bind and Spin DNA
Place a QIAquick spin column in a provided 2 mL collection tube.
Apply the sample to the QIAquick column and centrifuge for 00:01:00 .
Note
If volume exceeds 800 µL , add and centrifuge only 800 µL first, then add and centrifuge the remaining volume.
Discard flow-through and place the QIAquick column back into the same tube.
Add 500 µL Buffer QG to the QIAquick column and centrifuge for 00:01:00 . Discard flow-through and place back into the same tube.
Wash DNA
Wash DNA
Add 750 µL Buffer PE to QIAquick column and centrifuge for 00:01:00 . Discard flow-through and place column back into the same tube.
Elute DNA
Elute DNA
Place QIAquick column into a clean 1.5 mL centrifuge tube.
Add 50 µL Buffer EB or Nuclease Free Water to the center of the QIAquick membrane.
Note
For increased DNA concentration add 30 µL instead.
Let stand for 00:04:00 to increase DNA yield.
Centrifuge the column for 00:01:00 .
Discard the QIAquick column, and store DNA in microcentrifuge tube or aliquot to smaller volume.