GE Healthcare

illustra™MicroSpin ColumnsDNA Purification columns for buffer exchange and primer removal

Product booklet

Codes: S-200 27-5120-01 (50 columns) S-300 27-5130-01 (50 columns) S-400 27-5140-01 (50 columns)

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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

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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.

www.gehealthcare.com/nap

GE Healthcare UK Limited.Amersham Place, Little Chalfont,Buckinghamshire, HP7 9NA UK

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safety data sheet(s) and/or safety statement(s) for specific advice.

2.2. Storage All kit components should be stored at 4ºC

2.3. Expiry For expiry date please refer to outer packaging label.

2. Handling

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

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3. Components

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.

50 columns

50 tubes

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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 µl, 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

100 columns

50 columns

28903470

27-5340-01

GFX™ PCR DNA and gel band purifi cation kit

AutoSeq™ G-50

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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

50 columns

20 columns

50 columns

28903427

28903408

17-0853-01

27-5325-01

AutoSeq 96 G-50

ProbeQuant™ G-50 Micro Columns

NAP™-5 columns

MicroSpin G-25 columns

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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.

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Prepare column

Add sample

Elute

Proceed to other applications

An overview of the MicroSpin Column procedure is given below:

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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 µl 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%.

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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

PCR reaction and enzymatic DNA reaction purification(buffer exchange, de-salt)

PCR reaction and enzymatic DNA reaction purification(removal of excess primers prior to cloning)

Will notremoveenzyme*

S-200 25-50 µl

Will not remove enzyme*

S-400 25-50 µl

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Application Notes Recommended Reaction column volume

PCR reaction and enzymatic DNA reaction purification(removal of excess primers prior to other applications)

Unincorporated labelled nucleotide removal from a DNA labelling reaction**

* To remove enzyme from PCR or enzymatic reactions, use GFX PCR and gel band extraction kit.

** DNA must be at least 100 bp in length for a good recovery.

For DNA less than 100 bp in length, use ProbeQuant columns.

For removal of unincorporated nucleotides from oligonucleotides, use MicroSpin G-25 columns.

Exceptions exist to these guides:

• Use MicroSpin S-300 columns for removal of primers from PCR reactions <200 bp in length, regardless of the intended application

• Use MicroSpin S-400 columns to remove primers that are greater than 24 bases in length, regardless of the size of PCR product

Will notremoveenzyme*

S-300S-400

25-50 µl50-100 µl

S-200S-300S-400

25-50 µl50-75 µl75-100 µl

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5. Protocol5.1. Column preparation5.1.1. Resuspend the resin in the column by vortexing.

5.1.2. Loosen the cap one-quarter turn and snap off the bottom closure.

5.1.3. Place the column in a collection tube (supplied).

5.1.4. Pre-spin the column for 1 minute at 735 x g

Care: Use of the pulse button on centrifuge may over-ride speed setting.Notes: Use columns immediately after preparation to avoid drying out of the resin.

For a relative centrifugal force (RCF) of 735 x g, the corresponding revolutions per minute (RPM) can be calculated using the following formula:

where r = radius of the rotor in mm, measured from centre of spindle to bottom of rotor bucket.

e.g., if an RCF of 735 x g is required using a rotor with a radius of 73 mm, the corresponding RPM would be 3000.

R PM = 1000 X R CF /1.12r

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The table below shows the appropriate RPM for various microcentrifuges

Microcentrifuge Appropriate RPM for an RCF of 735 x gHeraeus Biofuge 15 2800Beckman GS15R 2100Hettich Mikro 24-48 2630Hettich Mikro EBA12 2700Eppendorf Centrifuge 5415C 3000Eppendorf Centrifuge 5417C 2700

5.2. Sample application5.2.1. Remove the top cap

5.2.2. Transfer the column to a new clean microcentrifuge tube. Slowly apply the sample to the centre of the resin bed.

Care: The resin will appear compacted and angled. Take care not to disturb the resin bed. Do not allow any of the sample to fl ow around the sides of the bed.

5.2.3. Spin the column for 2 minutes at 735 x g. Purifi ed sample is collected in the bottom of the microcentrifuge tube.

5.2.4. Remove the spin column from the microcentrifuge tube and discard.

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6. Appendices6.1. Troubleshooting guideThis guide may be helpful in the fi rst instance. If you need any further information, GE Healthcare Technical Services are always happy to assist. http://www.gehealthcare.com/nap

Problem Suggestions

Poor sample purity a) Ensure the sample volume was within acceptable range prior to loading (see application guide) b) Ensure sample is CAREFULLY pipetted into centre of resin. Do not disturb the column. Do not allow the sample to run into the sides of the resin bed. c) Use the column immediately after completing step 5.1 column preparation. Do not allow the resin to become dried out or cracked.

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6.2. Related products available from GE Healthcare

Product

AutoSeq G-50ExoSAP-IT™

NAP 5 columnsMicroSpin G-25 columns

GFX PCR DNA and gel band purifi cation kit

ProbeQuant G-50 Micro Columns

Rediprime™ II DNA labelling systemNick™ Translation kit5’-end labelling kitHybond™-N+Hybond™-XL

Pack Size

50 columns500 reactions

50 columns50 columns

100 columns

50 columns

30 reactions

20 reactions

20 reactions

Application

27-5340-01US78201

17-0853-0227-5325-01

28903470

28903408

RPN1633

N5000

RPN1509VariousVarious

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27512001 PL Rev D 2006

http://www.gehealthcare.com/nap

GE Healthcare UK LimitedAmersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK

imagination at work

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The next two pages are a protocol card.Please add to the back page as a tear off addition.

illustraMicroSpin ColumnsDNA purification columns for buffer exchange and primer removalExperienced user protocol

1.

2.

3.

4.

5.

6.

Resuspend resin by vortexing

Loosen cap and snap off bottom closure

Insert column into collection tube

Pre-spin

Insert colunm into 1.5 ml microcentrifuge tube

Carefully add sample

27-5120-0127-5130-0127-5140-01

1 minute at 735 x g

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8.

Spin

Discard column

2 minutes at 735 x g

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.

www.gehealthcare.com/nap

GE Healthcare UK Limited.Amersham Place, Little Chalfont,Buckinghamshire, HP7 9NA UK

imagination at work