illustra MicroSpin G-25 Columns Spin columns for the purification of oligonucleotides and small DNA fragments

Transcription

1 GE Healthcare illustra MicroSpin G-25 Columns Spin columns for the purification of oligonucleotides and small DNA fragments Product booklet Code: (50 columns)

2 Page finder 1. Legal 3 2. Handling Safety warnings and precautions Storage Expiry 4 3. Components Kit contents Reagents to be supplied by user 5 4. Description When to use a MicroSpin G-25 column The basic principle Guidelines for use 9 5. Protocol Column preparation Sample Application Appendices Troubleshooting guide Related products available from GE Healthcare 14 Tear off sheet Experienced user protocol 2

3 1. Legal Product use restriction The MicroSpin G-25 columns and 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 as the performance characteristics of this product has not been verified to a specific organism. GE and GE monogram are trademarks of General Electric Company AutoSeq, GFX, illustra, MicroSpin, NAP, NICK, ProbeQuant, Rediprime and Sephadex are trademarks of GE Healthcare companies Kathon is a trademark of Rohm and Haas Company 2006 General Electric Company All rights reserved. GE Healthcare reserves the right, subject to any regulatory and contractual approval, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE 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 3

4 2. Handling 2.1. Safety warnings and precautions Warning: For research use only. Not recommended or 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 safety data sheet(s) and/or safety statement(s) for specific advice Storage All kit components should be stored at room temperature (20-25 C) Expiry For expiry date please refer to outer packaging label. 4

5 3. Components 3.1. Kit contents Pack Size 50 Cat. No MicroSpin G-25 columns Collection tubes 50 columns 50 tubes MicroSpin G-25 columns equilibrated in double-distilled water, containing 0.05% Kathon CG/ICP Biocide as preservative Reagents to be supplied by user Clean microcentrifuge tube for the collection of the final eluted sample. 5

6 4. Description 4.1. When to use a MicroSpin G-25 column MicroSpin G-25 columns are designed for the rapid purification of DNA for use in a wide range of applications, including desalting, buffer exchange, and removal of unincorporated nucleotides from end-labelled oligonucleotides. They can be used for any DNA greater than 10 bases in length and are therefore ideal for the purification of oligonucleotides or very small DNA fragments following synthesis or a labelling reaction. They will not remove or denature enzyme. GE Healthcare provides a wide range of nucleic acid purification products, some of which may 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 Size Code PCR reaction and GFX PCR DNA enyzymatic DNA reaction and gel band columns purification 100 bp-10 kb purification kit size range (enzyme removal, buffer exchange, de-salt, primer removal) Extraction of DNA from agarose gels Dye terminator removal AutoSeq G columns from automated sequencing reactions AutoSeq 96 G x 96 well plates 6

7 Application Product Product Pack Size Code Unincorporated labelled ProbeQuant columns nucleotide removal G-50 Micro from a DNA labelling Columns reaction (>20 mers) Purification of NAP columns oligonucleotides Columns following synthesis (buffer exchange and de-salt). Gravity format, 500 μl loading volume 4.2. The basic principle MicroSpin G-25 columns contain sephadex resin. They allow DNA purification by the process of gel filtration. Molecules larger than the largest pores in the sephadex are excluded from the gel and elute first. Intermediate size molecules penetrate the matrix to varying extents, depending on their size and the resin used. 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 7

8 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. An overview of the MicroSpin G-25 column procedure is given below: Prepare column Add sample Elute Proceed to other applications 8

9 4.3. Guidelines for use MicroSpin G-25 columns can be used for a wide variety of DNA purificiation applications. The DNA to be purified must be at least 10 bases in length. When choosing the appropriate application, the following guidelines should be considered: 20x Rule The best results will be obtained when the product being purified 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 Non-specific binding The non-specific binding exhibited by the MicroSpin columns is relatively insignificant, allowing purification of samples in the nanogram range. 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 volumes Load μl onto column when desalting oligonucleotides following deprotection in ammonia. For larger volumes (500 μl), consider use of our NAP 5 columns; see table section 6.2. for ordering details. Load μl onto column for removal of unincorporated labelled nucleotides from oligonucleotide labeling reactions. For DNA fragments greater than 20 bases, we recommend use of ProbeQuant G-50 columns; see table in section 6.2. for ordering details. MicroSpin G-25 columns are optimised for purification of 9

10 small DNA fragments. Load 50 μl onto column for buffer exchange or de-salt of DNA. For purification 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 enzyme removal is important to your downstream application and you are using a MicroSpin G-25 column for very small DNA fragments, you must phenol chloroform extract prior to loading onto the column. Phenol:chloroform:isoamyl alcohol is available from GE Healthcare (catalogue number US75831). For sample volumes larger than 50 μl, 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. Nuclease Testing MicroSpin G-25 columns are tested in nickase, single and double-stranded exonuclease and RNase assays. 10

11 5. Protocol 5.1. Column preparation 1. Resuspend the resin in the column by vortexing. 2. Loosen the cap one-quarter turn and snap off the bottom closure. 3. Place the column in a collection tube (supplied). 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 the corresponding revolutions per minute (RPM) can be calculated using the following formula: R PM = 1000 X R CF /1.12r 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

12 The table below shows the appropriate RPM for various microcentrifuges Microcentrifuge Appropriate RPM for an RCF of 735 x g Heraeus Biofuge Beckman GS15R 2100 Hettich Mikro Hettich Mikro EBA Eppendorf Centrifuge 5415C 3000 Eppendorf Centrifuge 5417C Sample Application 1. Remove the top cap 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 flow around the sides of the bed. 3. Spin the column for 2 minutes at 735 x g Purified sample is collected in the bottom of the microcentrifuge tube. 4. Remove the spin column from the microcentrifuge tube and discard 12

13 6. Appendices 6.1. Troubleshooting guide This guide may be helpful in the first instance. If you need any further information, GE Healthcare technical services are always happy to assist. Problem Poor sample purity Suggestions a) Ensure the sample volume was within acceptable range prior to loading (see usage guidelines) 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. 13

14 6.2. Related products available from GE Healthcare Product number Product Pack Size AutoSeq G columns AutoSeq 96 G x 96 well plates US78201 ExoSAP-IT 500 reactions NAP-5 columns 50 columns GFX PCR DNA and gel band purification kit ProbeQuant G-50 micro columns 100 columns 50 columns RPN1633 Rediprime II DNA labelling system 30 reactions N5000 Nick Translation kit 20 reactions RPN end labelling kit 20 reactions 14

15 15

16 GE Healthcare offices: GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D Freiburg Germany GE Healthcare regional office contact numbers: Asia Pacific Tel: Fax: Australasia Tel: Fax: France Tel: Fax: Germany Tel: Fax: Greater China Tel: Fax: Portugal Tel: Fax: Russia & other C.I.S. & N.I.S Tel: Fax: Spain Tel: Fax: GE Healthcare UK Limited Amersham Place Little Chalfont Austria Tel: 01/ Fax: 01/ Italy Tel: Fax: Sweden Tel: Fax: Buckinghamshire HP7 9NA UK Belgium Tel: Fax: Japan Tel: Fax: Switzerland Tel: Fax: GE Healthcare Bio-Sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway NJ USA GE Healthcare Bio-Sciences KK Sanken Bldg Hyakunincho Shinjuku-ku Tokyo Japan Canada Tel: Fax: Central & East Europe Tel: Fax: Denmark Tel: Fax: Eire Tel: Fax: Korea Tel: Fax: Latin America Tel: Fax: Middle East & Africa Tel: Fax: Netherlands Tel: Fax: UK Tel: Fax: USA Tel: Fax: Finland & Baltics Norway Tel: +358-(0) Tel: Fax: +358 (0) Fax: GE Healthcare UK Limited Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK imagination at work PL Rev A 2006

17 The next two pages are a protocol card. If required please add to the back page as a tear off addition If not then delete these three pages.

18 illustra MicroSpin G-25 Columns Spin columns for the purification of oligonucleotides and small DNA fragments Experienced user protocol 1. Resuspend resin by vortexing 2. Loosen cap and snap off bottom closure 3. Insert column into collection tube 4. Pre-spin 1 minute at 735 x g 5. Insert column into 1.5 ml microcentrifuge tube 6. Carefully add sample PC Rev A 2006

19 MicroSpin G-25 Columns Spin columns for the purification of oligonucleotides and small DNA fragments 7. Spin 2 minutes at 735 x g 8. Discard column GE and GE monogram are trademarks of General Electric Company. illustra and MicroSpin are trademarks of GE Healthcare companies General Electric Company All rights reserved. GE Healthcare reserves the right, subject to any regulatory approval, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE 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 imagination at work