Dec 20, 2016
Zymoclean™ Gel DNA Recovery Kit
- Zymo Research
- Zymo Research - The Epigenetics Company
External link: https://www.zymoresearch.com/dna/dna-clean-up/gel-dna-recovery/zymoclean-gel-dna-recovery-kit
Protocol Citation: Zymo Research 2016. Zymoclean™ Gel DNA Recovery Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.gitbuen
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: November 25, 2016
Last Modified: March 25, 2018
Protocol Integer ID: 4403
Abstract
Highlights
Quick (15 minute) high-yield recovery of ultra-pure DNA from agarose gels.
Column design permits DNA elution at high concentrations into minimal volumes (≥ 6 μl).
Eluted DNA is well suited for use in DNA ligation, sequencing, labeling, PCR, etc.
Guidelines
Product Contents
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.
Buffer Preparation
Before starting: 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.
Troubleshooting
Low Recovery
Ensure Agarose is Fully Dissolved
There may be small globules of undissolved agarose in the sample that can interfere with DNA recovery by clogging the column and leeching salts into the eluate.
Gel Dissolved at Temperatures Above 60 °C
If dissolved at a higher temperature, DNA may be denatured affecting recovery. For optimal results, dissolve the gel slice between 37-55 °C.
Improperly Prepared/Stored DNA Wash Buffer
Make sure ethanol has been added to the DNA Wash Buffer concentrate. Cap the bottle tightly to prevent evaporation over time.
Addition of DNA Elution Buffer
Add elution buffer directly to the column matrix, not to the walls of the column. Elution buffer requires contact with the matrix for at least 1 minute for large DNA ≥ 10kb.
Incomplete Elution
DNA elution is dependent on pH, temperature, and time. For large genomic DNA (≥ 50 kb), apply heated elution buffer (60-70 °C) to the column and incubate for several minutes prior to elution.
Sequential elutions may be performed for quantitatively higher recovery but lower final DNA concentration. This is recommended for DNA ≥ 10 kb.
Low A260/A230 ratio
Column tip contaminated
When removing the column from the collection tube, be careful that the tip of the column does not come into contact with the flowthrough. Trace amounts of salt from the flowthrough can contaminate a sample resulting in a low A260/A230 ratio. Ethanol contamination from the flowthrough can also interfere with DNA elution. Zymo-Spin™ columns are designed for complete elution with no buffer retention or carryover (see below).
Following Clean-up with the DCCTM, Multiple Bands Appear in an Agarose Gel
Acidification of DNA Loading Dye
Most loading dyes do not contain EDTA and will acidify (pH ≤ 4) over time due to some microbial growth. This low pH is enough to cause DNA degradation. Therefore, if water is used to elute the DNA, 6X Loading Dye containing 1 mM EDTA is recommended.
Materials
MATERIALS
Zymoclean™ Gel DNA Recovery KitZymo ResearchCatalog #D4001
Safety warnings
All centrifugation steps should be performed between 10,000 - 16,000 x g.
Before start
Before starting: 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.
Step
Step
Excise the DNA fragment1 from the agarose gel using a razor blade, scalpel or other device and transfer it into a 1.5 ml microcentrifuge tube.
Note
1 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 (e.g. for 100 μl (mg) of agarose gel slice add 300 μl of ADB).
Incubate at 37-55 °C for 5-10 minutes until the gel slice is completely dissolved2.
00:10:00
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
2 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.
Transfer the melted agarose solution to a Zymo-Spin™ Column in a Collection Tube.
Centrifuge for 30-60 seconds. Discard the flow-through3.
00:01:00
Note
3 Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.
Note
All centrifugation steps should be performed between 10,000 - 16,000 x g.
Add 200 μl of DNA Wash Buffer to the column and centrifuge for 30 seconds. Discard the flow-through. (wash 1/2)
00:00:30
Add 200 μl of DNA Wash Buffer to the column and centrifuge for 30 seconds. Discard the flow-through. (wash 2/2)
00:00:30
Add ≥ 6 μl DNA Elution Buffer4 or water5 directly to the column matrix.
Note
4 DNA Elution Buffer: 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA.
Note
5 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 ºC DNA Elution Buffer.
Place column into a 1.5 ml tube and centrifuge for 30-60 seconds to elute DNA.
00:01:00
Note
All centrifugation steps should be performed between 10,000 - 16,000 x g.
Ultra-pure DNA is now ready for use.