Jul 14, 2015
Zymoclean™ Gel DNA Recovery Kit
- Alan J. Cone1
- 1Wright State University
- Ju Lab
Protocol Citation: Alan J. Cone 2015. Zymoclean™ Gel DNA Recovery Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.ddc22v
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
Created: July 13, 2015
Last Modified: March 16, 2018
Protocol Integer ID: 1156
Abstract
This is a protocol for high yield recovery of pure DNA from agarose gels using the Zymoclean™ Gel DNA Recovery Kit.
Guidelines
All centrifugation steps should be performed between 10,000 - 16,000 x g.
Product Contents
| Zymoclean™ Gel DNA Recovery Kit (Kit Size) | D4001, D4007 (50 Preps.) | D4002, D4008 (200 Preps.) | Storage Temperature | |
| ADB | 50 ml | 2x100 ml | Room Temp. | |
| DNA Wash Buffer1 | 6 ml | 24 ml | Room Temp. | |
| DNA Elution Buffer | 1 ml | 4 ml | Room Temp. | |
| Zymo-Spin™ I Columns | 50 D4001 – uncapped columns D4007 – capped columns | 200 D4002 – uncapped columns D4008 – capped columns | Room Temp. | |
| Collection Tubes | 50 | 200 | Room Temp. | |
| Instruction Manual | 1 | 1 | - |
Integrity of kit components is guaranteed for up to one year from date of purchase. Reagents are routinely tested on a lot-to-lot basis to ensure they provide the highest performance and reliability.
1 Ethanol must be added prior to use as indicated on the DNA Wash Buffer label.
Specifications
• DNA Purity – High-quality, purified DNA is especially well suited for sequencing and ligation reactions.
• DNA Size Limits – From ~50 bp to 23 kb.
• DNA Recovery – Typically, up to 5 µg total DNA per column can be eluted into as little as 6 µl of low salt DNA Elution Buffer or water. For DNA 50 bp to 10 kb, the recovery is 70-90%. For DNA 11 kb to 23 kb, the recovery is 50-70%.
• Sample Sources – DNA in excised agarose gel slices.
• Product Detergent Tolerance – ≤ 5% Triton X-100, ≤ 5% Tween-20, ≤ 5% Sarkosyl, ≤ 0.1% SDS.
Product Description
The Zymoclean™Gel DNA Recovery Kit provides a hassle-free method for high yield recovery of pure DNA from agarose gels. Simply add the specially formulated Agarose Dissolving Buffer (ADB) to the gel slice containing your DNA sample, let dissolve, and then transfer to the supplied Zymo-Spin™ Column. There is no need for organic denaturants or chloroform. Instead, the product utilizes Fast-Spin column technology to yield high-quality DNA in just 15 minutes (See figures below). DNA purified using the Zymoclean™ Gel DNA Recovery Kit is perfectly suited for use in DNA ligation reactions, sequencing, DNA labeling reactions, PCR, etc.
Zymoclean™ products are offered in single column (uncapped or capped column) or 96-well format. In addition, the Zymoclean™ Large Fragment DNA Recovery Kit is designed for large DNA (up to 200 kb) gel recovery.
Materials
MATERIALS
ADB (Agarose Dissolving Buffer)Catalog #D4001-1-50
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Zymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1
Zymoclean™ Gel DNA Recovery Kit - Uncapped columns Zymo ResearchCatalog #D4001
STEP MATERIALS
ADB (Agarose Dissolving Buffer)Catalog #D4001-1-50
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Zymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1
ADB (Agarose Dissolving Buffer)Catalog #D4001-1-50
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Zymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1
Protocol materials
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Zymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1
Zymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1
ADB (Agarose Dissolving Buffer)Catalog #D4001-1-50
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Zymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1
Zymoclean™ Gel DNA Recovery Kit - Uncapped columns Zymo ResearchCatalog #D4001
ADB (Agarose Dissolving Buffer)Catalog #D4001-1-50
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
ADB (Agarose Dissolving Buffer)Catalog #D4001-1-50
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
ADB (Agarose Dissolving Buffer)Catalog #D4001-1-50
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Zymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1
Before start
Add 24 ml 100% ethanol (26 ml 95% ethanol) to the 6 ml DNA Wash Buffer concentrate. Add 96 ml 100% ethanol (104 ml 95% ethanol) to the 24 ml DNA Wash Buffer concentrate.
Excise the DNA fragment from the agarose gel using a razor blade, scalpel or other device and transfer it into a 15 ml microcentrifuge tube
Note
The amount of agarose excised from the gel should be as small as possible.
Add 3 volumes of ADB to each volume of agarose excised from the gel.
Note
(e.g. for 100 µl (mg) of agarose gel slice add 300 µl of ADB)
ADB (Agarose Dissolving Buffer)Catalog #D4001-1-50
Incubate at 37-55 °C for 5-10 minutes until the gel slice is completely dissolved.
00:10:00
Note
Do not incubate above 60°C. It is important that the gel slice dissolve completely. This can be facilitated by gentle mixing during the incubation.
Note
For DNA fragments > 8 kb, following the incubation step, add one additional volume (equal to that of the gel slice) of water to the mixture for better DNA recovery (e.g., 100 µl agarose, 300 µl ADB, and 100 µl water).
Note
I like to let the agarose dissolve for 15 minutes at 55 °C versus the Zymo recommendation.
Transfer the melted agarose solution to a Zymo-Spin™ Column in a Collection Tube.
Centrifuge for 30-60 seconds. Discard the flow-through.
00:01:00
Note
Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.
Wash #1:Add 200 µl of DNA Wash Buffer to the column.
200 µL
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Wash #1:Centrifuge for 30 seconds. Discard the flow-through.
00:00:30
Wash #2: Add 200 µl of DNA Wash Buffer to the column.
200 µL
Note
Ultra-pure DNA is now ready for use.
Zymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6
Wash #2:Centrifuge for 30 seconds. Discard the flow-through.
00:00:30
Note
DNA Elution Buffer: 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA.
Elution of DNA from the column is dependent on pH and temperature. If water is used, make sure the pH is >6.0. Waiting 1 minute prior to elution may improve the yield of larger (> 6 kb) DNA. For even larger DNA (> 10 kb), the total yield may be improved by eluting the DNA with 60-70 o C DNA Elution Buffer
Add ≥ 6 µl DNA Elution Buffer or waterdirectly to the column matrix.
6 µL
Note
I use exactly 6 uL of their elution buffer.
Zymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1
Place column into a 1.5 ml tube and centrifuge for 30-60 seconds to elute DNA.
00:01:00