Dec 21, 2016
DNA Clean & Concentrator™-5 Protocol
- Zymo Research
- Zymo Research - The Epigenetics Company
External link: http://www.zymoresearch.com/dna/dna-clean-up/pcr-dna-clean-up-concentration/dna-clean-concentrator-5
Protocol Citation: Zymo Research 2016. DNA Clean & Concentrator™-5 Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.giubuew
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 28, 2018
Protocol Integer ID: 4404
Abstract
The DNA Clean & Concentrator™-5 (DCC™-5) provides a hassle-free method for the rapid purification and concentration of high-quality DNA from PCR, endonuclease digestions, cell lysates, and other impure DNA preparations. It can also be used for post-RT cDNA clean-up and purification of sequencing-ready DNA from M13 phage.
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 DNA (A260/A280 >1.8) ideal for ligation, sequencing, labeling, PCR, microarray, transfection, transformation, and restriction digestion procedures.
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 from enzymatic reactions (e.g., PCR, restriction endonuclease digestions), plasmid preparations, and impure preparations.
Product Detergent Tolerance – ≤5% Triton X-100, ≤5% Tween-20, ≤5% Sarkosyl, ≤ 0.1% SDS.
Product Description
The DNA Clean & Concentrator™-5 (DCC™-5) provides a hassle-free method for the rapid purification and concentration of high-quality DNA from PCR, endonuclease digestions, cell lysates, and other impure DNA preparations. It can also be used for post-RT cDNA clean-up and purification of sequencing-ready DNA from M13 phage. Simply add the specially formulated DNA Binding Buffer to your sample and transfer the mixture to the supplied Zymo-Spin™ Column. There is no need for organic denaturants or chloroform. Instead, the product features Fast-Spin column technology to yield DNA that is free of salts and contaminants in just 2 minutes. The purified DNA is ideal for DNA ligation, sequencing, labeling, PCR, microarray, transfection, transformation, and restriction digestion procedures.
Available Formats
Typical DCC™ Applications
For purification of short DNA or RNA oligonucleotides ≥ 16 nt, use the Oligo Clean & Concentrator (D4060, D4061).
For ChIP (Chromatin Immunoprecipitation) sample cleanup, use the ChIP DNA Clean & Concentrator (D5201,D5205) for high quality DNA from any step in a standard ChIP protocol.
For post-cycle sequencing samples, use the ZR Sequencing DNA Clean-up Kit (D4050, D4051) for dye blob elimination.
For samples containing PCR inhibitors, use the OneStep™ PCR Inhibitor Removal Kit (D6030, D6035).
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.
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Troubleshooting
Low Recovery
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.
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Selected Citations
Li, N. (2010). Whole genome DNA methylation analysis based on high throughput sequencing technology. Methods, 52 (3), 221-232.
Lee, EJ. (2011). Targeted bisulfite sequencing by solution selection and massively parallel sequencing. Nucleic Acids Research, 39(19),
e127, doi:10.1093/nar/gkr598
Papageorgiou, EA. (2009). Sites of differential DNA methylation between placenta and peripheral blood. Am J Pathol, 174 (5), 1609-1618.
Ferguson, A.A. et al. (2009). Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment. Plasmid, 62, 140-145.
Materials
MATERIALS
DNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003
Before start
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.
In a 1.5 ml microcentrifuge tube, add 2-7 volumes of DNA Binding Buffer to each volume of DNA sample (see table below). Mix briefly by vortexing.
For efficient recovery of genomic or large DNA (> 20 kb to > 200 kb), use the Genomic DNA Clean & Concentrator™ (Cat. Nos. D4010, D4011).
Note
Transfer mixture to a provided Zymo-Spin™ Column2 in a Collection Tube.
Note
2 The sample capacity of the column is 800 μl. Therefore, it may be necessary to load and spin a column multiple times if a sample has a volume larger than 800 μl.
Centrifuge for 30 seconds. Discard the flow-through.
00:00:30
Add 200 μl DNA Wash Buffer to the column. Centrifuge for 30 seconds. (wash 1/2)
00:00:30
Add 200 μl DNA Wash Buffer to the column. Centrifuge for 30 seconds. (wash 2/2)
00:00:30
Add ≥ 6 μl DNA Elution Buffer3 or water4 directly to the column matrix and incubate at room temperature for one minute.
00:01:00
Note
3 DNA Elution Buffer: 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA
Note
4 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 °C DNA Elution Buffer.
Transfer the column to a 1.5 ml microcentrifuge tube and centrifuge for 30 seconds to elute the DNA.
00:00:30
Note
Ultra-pure DNA is now ready for use.