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illustraMicroSpin ColumnsDNA Purification columns for buffer exchange and primer removal
Codes: S-200 27-5120-01 (50 columns) S-300 27-5130-01 (50 columns) S-400 27-5140-01 (50 columns)
Page finder 1. Legal 4
2. Handling 5 2.1. Safety warnings and precautions 5 2.2. Storage 5 2.3. Expiry 5
3. Components 6 3.1. Kit contents 6 3.2. Materials to be provided by user 6
4. Description 74.1. When to use a MicroSpin S-200, S-300 or S-400 column 74.2. The basic principle 94.3. General guidelines for use 11 4.4. Column specific guidelines 12
5. Protocols 145.1. Column preparation 145.2. Sample application 15
6. Appendices 166.1. Troubleshooting guide 166.2. Related products available from GE Healthcare 17
Tear off sheet Experienced user protocol
1. Legal Product use restrictionThe MicroSpin range components have been designed, developed and sold for research purposes only. They are suitable for in vitro use only. No claim or representation is intended for its use to identify any specific organism or for clinical use (diagnostic, prognostic, therapeutic, or blood banking).
It is the responsibility of the user to verify the use of MicroSpin columns for a specific application range.
GE and GE monogram are trademarks of General Electric Company. MicroSpin, Sephacryl, GFX, AutoSeq, NAP, ProbeQuant, NICK, Hybond, Rediprime and illustra are trademarks of GE Healthcare companies.ExoSAP-IT is a trademark of USB Corp.
2006 General Electric Company All rights reserved.
GE Healthcare reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation.Contact your GE Healthcare representative for the most current information and a copy of the terms and conditions.
GE Healthcare UK Limited.Amersham Place, Little Chalfont,Buckinghamshire, HP7 9NA UK
safety data sheet(s) and/or safety statement(s) for specific advice.
2.2. Storage All kit components should be stored at 4C
2.3. Expiry For expiry date please refer to outer packaging label.
2.1. Safety warnings and precautions Warning: For research useonly. Not recommendedor intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material
3.1. Kit contents
Pack Size 50Cat. No. 27-5120-01MicroSpin S-200 HR columns, pre-packed with Sephacryl S-200 HR resin and equilibrated in TE buffer (pH 7.6)Collection tubes
Pack Size 50Cat. No. 27-5130-01MicroSpin S-300 HR columns, 50 columnspre-packed with Sephacryl S-300 HR resin and equilibrated in TE buffer (pH 7.6)Collection tubes 50 tubes
Pack Size 50Cat. No. 27-5140-01MicroSpin S-400 HR columns, 50 columnspre-packed with Sephacryl S-400 HR resin and equilibrated in TE buffer (pH 7.6)Collection tubes 50 tubes
3.2. Materials to be supplied by userClean microcentrifuge tube for the collection of the fi nal eluted sample.
4. Description4.1. When to use a MicroSpin S-200, S-300 or S-400 columnMicroSpin columns are designed for the rapid purifi cation of nucleic acids for use in a wide range of applications, including desalting, buffer exchange and removal of primers. Good product yield and purity is obtained with sample volumes from 25-100 l, and from nanogram to milligram quantities of DNA.
GE Healthcare provides a wide range of nucleic acid purifi cation products, some of which might be better suited to your application. These products and the application for which they have been optimised are summarised in the table below.
Application Product Product Pack Code Size
PCR reaction and enzymatic DNA reaction purifi cation(enzyme removal, buffer exchange, de-salt, primer removal), 100 bp-10 kb size rangeExtraction of DNA from agarose gels
Dye terminator removalfrom automated sequencing reactions
GFX PCR DNA and gel band purifi cation kit
Application Product Product Pack Code Size
Dye terminator removal from automated sequencing reactions (96 well format)
Unincorporated labelled nucleotide removalfrom a DNA labelling reaction
Purifi cation of oligonucleotides following synthesis (buffer exchange and de-salt). Gravity format
Purifi cation of oligonucleotides following synthesis (buffer exchange and de-salt). Spin column format
10 x 96 well plates
AutoSeq 96 G-50
ProbeQuant G-50 Micro Columns
MicroSpin G-25 columns
4.2. The basic principleMicroSpin columns contain Sephacryl resin of differing pore sizes. They allow rapid DNA purification by the process of gel filtration. Molecules larger than the largest pores in the Sephacryl are excluded from the gel and elute first. Intermediate size molecules penetrate the matrix to varying extents, depending on their size. Penetration of the matrix retards progress through the column; very small molecules elute last. The volume required to elute these small molecules is dependent on the volume available both inside and outside the pores i.e. the bed volume.
Gel filtration resins do not exhibit a fixed exclusion limit when used in a spin-column format. Exclusion limits of gel filtration resins are only meaningful in continuous flow processes where the molecules being purified have sufficient time to reach equilibrium between the time spent in the gel filtration medium and the time spent in the eluent stream. In spin-column chromatography, the observed exclusion properties that allow the product to pass through the gel while the smaller impurities are retained depends on experimental factors, such as: the resin used, sample volume, product size, and the g forces used in the purification process.
Proceed to other applications
An overview of the MicroSpin Column procedure is given below:
4.3. General guidelines for useMicroSpin Sephacryl HR columns can be used for a wide variety of DNA purifi cation applications. When choosing the appropriate column for your application, the following general guidelines should be considered:
20x Rule The best results will be obtained when the product that is being purifi ed is at least 20 times larger than the largest impurity. If the difference in size is less than 20-fold, either purity or yield may be compromised
Purity versus yield In general, purity is inversely proportional to yield. Larger sample volumes will provide higher yield but lower purity, and vice-versa. For any given volume, the larger the pore size of the resin, the greater the purity and lower the yield of the product which results. Gel fi ltration matrices with larger pore sizes (SephacrylS-400>Sephacryl S-300>Sephacryl S-200) tend to retain more product than gel matrices with smaller pores.
Non-specifi c binding The non-specifi c binding exhibited by the MicroSpin columns is relatively insignifi cant, allowing purifi cation of samples in the nanogram range. For each resin type there will be a uniform proportional loss of sample which is due to the nature of spin column chromatography.
Retention For a given sample volume, product retention is relative to molecular size. As the size of the product increases, its relative retention decreases.
Loading Volume Load 25-100 l onto a column for all applications. For larger sample volumes, either use more than one column or reduce the sample volume by drying or precipitation. For smaller sample volumes, dilute the sample to improve product recovery.
If the volume recommendations for the following applications are followed, the yield of purifi ed DNA is expected to be 50-90%.
Enzyme RemovalFor purifi cation of DNA fragments 100 bp - 10 kb in length, following an enzymatic reaction, we recommend using the GFX PCR and gel extraction kit, as the enzyme will be removed during the spin column process. If using a MicroSpin column, you must phenol chloroform extract prior to loading onto the column to ensure enzyme removal. Phenol:chloroform:isoamyl alcohol is available from GE Healthcare (catalogue number US75831).
Nuclease Testing MicroSpin Sephacryl HR columns are tested in nickase, single and double-stranded exonuclease and RNase assays.
4.4. Column specific guidelinesFor more specific column selection, a simplified applications guide is given below
Application Notes Recommended Reaction Column volume