Page finder. 1. Legal Handling Safety warnings and precautions Storage Expiry 4

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1 GE Healthcare illustra ProbeQuant G-50 Micro Columns Radiolabeled Probe Purification Kit (For the removal of unincorporated labeled nucleotides from DNA labeling reactions) Product booklet Code: (50 columns)

2 Page finder 1. Legal 3 2. Handling Safety warnings and precautions Storage Expiry 4 3. Components Kit contents Materials to be supplied by user Equipment to be supplied by user 6 4. Description The basic principle Kit specifications Protocol Purification of Sample Determination of cpm/ul of Labeled probe Determination of Percent Incorporation of Radiolabel Appendices Troubleshooting guide Related products available from GE Healthcare 21 Tear off sheet Experienced user protocol 2

3 1. Legal Product use restriction The ProbeQuant TM G-50 Micro Columns 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 the ProbeQuant G-50 Micro Columns for a specific application range as the performance characteristic of this kit has not been verified to a specific organism. GE and GE monogram are trademarks of General Electric Company AutoSeq, CyScribe, GFX, Hybond, Hyperfilm, illustra, Megaprime, MicroSpin, NAP, NICK, ProbeQuant, Ready-To-Go, Rapid-hyb, Rediprime, Redivue, 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, 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 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. data sheet(s) and/or safety statement(s) for specific advice Storage Store at ambient temperature Expiry For expiry date please refer to outer packaging label. 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 4

5 3. Components 3.1. Kit contents Pack Size 50 Cat. No ProbeQuant G-50 Micro columns 50 columns Probe Elution and Storage Buffer (150 mm STE buffer, ph 8.0) 10 ml Collection tubes 50 tubes ProbeQuant G-50 Micro columns contain Sephadex G-50 DNA Grade F. Columns are supplied pre-equilibrated in 150 mm STE buffer containing 0.15% Kathon CG/ICP Biocide. 5

6 3.2. Materials to be supplied by user Recipe for additional Probe Elution and Storage Buffer (150 mm STE buffer, ph 8.0) To prepare 50 ml of buffer, dissolve g of NaCl in 40 ml of TE buffer (10 mm Tris-HCl, ph 8.0, and 1 mm EDTA) with stirring. Titrate the solution to ph 8.0 using dilute HCl or NaOH, as required. Transfer the solution to a 50 ml graduated cylinder and adjust the volume to 50 ml with TE buffer, ph Equipment to be supplied by user Microcentrifuge. 1.5 ml microcentrifuge tubes Scintillation vials or microcentrifuge tube holders Scintillation counter 6

7 4. Description ProbeQuant G-50 Micro columns are designed for the quantitative removal of unincorporated labeled nucleotides from a DNA labeling reaction (labeled DNA should be at least 20 bases in length). They can be used for both preparative and analytical applications simultaneously. Columns can be used for the following: Purification of a DNA labeling reaction prior to applications such as hybridization. Determination of the cpm/μl of labeled probe. Measurement of the percent incorporation of a radionucleotide precursor into a labeled probe. Note: ProbeQuant G-50 Micro columns are intended for single-use only. ProbeQuant G-50 Micro columns are ready-to-use, require less than 4 minutes from sample application to collection of purified product. These columns provide a pre-packed and pre-equilibrated alternative to TCA precipitations. The sample elutes in 150 mm NaCl. RNA probes and biotinylated probes: Although tests have shown low levels of RNase activity for these columns, they are not specifically treated to be RNase free. Customers have regularly used our ProbeQuant columns for purification of RNA and are very satisfied. However, as the columns 7

8 have not been tested for absence of RNase s we cannot guarantee that the RNA will not be degraded - but in principle it works well. Biotinylated probes can be purified using the ProbeQuant columns (as it is the size of the probe, and not the modification that is relevant). Nuclease Testing: Tested in nickase, single and double-stranded exonuclease and RNase assays. For purification of labeled DNA less than 20 bases in length: We recommend use of our MicroSpin G-25 columns (product number: ). These columns are suitable for removal of unincorporated labeled nucleotides from end-labeled oligonucleotides at least 10 bases long. Please note that these columns are supplied in double-distilled water containing 0.05% Kathon and would need to be equilibrated in 150 mm STE buffer ph 8.0 before use The basic principle ProbeQuant G-50 Micro columns contain Sephadex G-50 DNA grade F. They are designed for the quantitative removal of unincorporated labeled nucleotides from a DNA labeling reaction 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. Penetration of the matrix retards progress through the column; very small molecules elute last. 8

9 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 an 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. The protocol provided with the ProbeQuant G-50 Micro columns has been optimised for the quantitative removal of unincorporated labeled nucleotides from a DNA labeling reaction only. GE Healthcare provide a range of nucleic acid purification products for other applications (see section 6.2. Related products available from GE Healthcare for more details). 9

10 The figure below outlines the basic steps in the simple purification of radiolabeled probes. Perform labeling reaction (see section 6.2. for range of labeling products available from GE Healthcare) Adjust final volume to 50 μl with Probe Elution and Storage Buffer (if required) Prepare column Add 50 μl sample Elute Purified Probe 10

11 4.2. Kit specifications Format Principle Column matrix Column buffer Maximum sample volume per column Yield / Recovery of DNA Length of labeled DNA recovered Major subsequent application Column capacity (maximum amount of DNA that can be loaded onto column) Spin-column. The principle of purification of the ProbeQuant column is gel filtration. This means that substances smaller than 1 x 10³ Da (or 20 bp) are retained in the matrix whereas larger molecules are eluted. Sephadex G-50 DNA grade 150 mm STE containing 0.15% Kathon 50 μl >80% >20 bp (N.B. there is no limitation on maximum length of probe that can be purified). Hybridization 10 μg 11

12 5. Protocol 5.1. Purification of Sample Perform the radiolabeling reaction according to standard instructions. Note: If percent label incorporation is to be determined, reserve 2 μl of the radiolabeling reaction for analysis. This is the TOTAL sample (see step of Determination of Percent Incorporation of Radiolabel ). Note: see section 6.2. Related Products for details of labeling products available from GE Healthcare Adjust the reaction volume to 50 μl with Probe Elution and Storage Buffer. Note: If the reaction volume is less than 50 μl, adjust to 50 μl with Probe Elution and Storage Buffer. If the reaction volume is greater than 50 μl, apply a 50 μl aliquot to a single column or use more than one column for purification. If more than 50 μl is loaded onto each column, unincorporated labeled nucleotides may elute off (see section 4.2. for basic principles of Sephadex) Resuspend the resin in the column by vortexing Loosen the cap one-quarter turn and snap off the bottom closure Place the column in the supplied collection tube for support. 12

13 Pre-spin the column for 1 minute at 735 x g (see Note below for help to calculate RPM from RCF). Note: Use columns immediately after preparation to avoid drying out of the resin. Note: if the column resin appears dry, displaced or cracked after the first spin, this is usually indicative of over-centrifuging. Rehydrate with 250 μl of Probe Elution and Storage Buffer, vortex and re-centrifuge at the correct settings. If the centrifugation was performed with the correct settings, reduce the speed of the centrifuge by 20%. Do not use the pulse button on the microcentrifuge as this may over-ride the speed setting. Note: For a relative centrifugal force (RCF) of 735 x g, the corresponding revolutions per minute (RPM) can be calculated using the following formula: RPM = 1000 X RCF/1.12r where r = radius of the rotor in mm, measured from the 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

14 The table below shows appropriate RPM for various microcentrifuges Microcentrifuge Appropriate RPM for an RCF of 735 x g Heraeus Biofuge rpm Beckman GS15R 2100 rpm Hettich Mikro rpm Hettich Mikro EBA rpm Eppendorf Centrifuge 5415C 3000 rpm Eppendorf Centrifuge 5417C 2700 rpm Place the column in a new 1.5 ml microcentrifuge tube and slowly apply 50 μl of the sample to the top-center of the resin, being careful not to disturb the resin bed. Note: after the pre-spin the resin will come away from the tube slightly to form a column within the tube, it is essential that the sample being purified is added slowly onto the top of the resin as shown in the diagram below. Make sure the solution does not run into the sides of the resin bed. Avoid touching the resin bed with the pipette tip. Figure showing technique for adding sample to column resin Spin the column at 735 x g for 2 minutes. The purified sample is collected in the bottom of the 1.5 ml tube Cap the collection tube. This is the COLUMN sample. Proceed to section 5.2. to determine the cpm/μl of labeled probe, or section 5.3. to determine the percent incorporation of the radiolabel. 14

15 5.2. Determination of cpm/μl of Labeled Probe Mix 2 μl of the COLUMN sample with 98 μl of STE buffer in a microcentrifuge tube Cap the tube and gently vortex to mix. Collect sample in bottom of microcentrifuge tube by pulse centrifugation Dispense 50 μl aliquots of the diluted sample into duplicate microcentrifuge tubes Transfer each tube to a scintillation vial or suitable tube holder for placement into the scintillation counter. Alternatively, the samples may be added directly to scintillation vials containing scintillant, capped and inverted to mix Count the samples using an appropriate program. Note: If no scintillant was added, 32 P-labeled samples may be counted using a 1 minute Cerenkov counting program; samples mixed with scintillant may be counted using a standard 32 P counting program Add the cpm obtained for the duplicate samples and divide by two; this value is the cpm/μl of the labeled probe. Note: This value represents the average cpm, as well as the cpm/μl of purified, labeled DNA. To determine the total cpm incorporated into the purified sample, multiply cpm/μl by the volume of the sample recovered from the column. 15

16 5.3. Determination of Percent Incorporation of Radiolabel For each sample to be assayed, mix 2 μl of the COLUMN sample with 98 μl of STE buffer in a microcentrifuge tube Prepare an identical dilution using the 2 μl sample reserved prior to column purification (see step of Sample Preparation ). This sample represents the TOTAL cpm added to the labeling reaction Cap the 4 tubes and gently vortex to mix. Collect sample in bottom of microcentrifuge by pulse centrifugation Dispense 50 μl aliquots of the diluted COLUMN and TOTAL samples into 4 microcentrifuge tubes Transfer each tube to a scintillation vial or suitable tube holder for placement into the scintillation counter. Alternatively, the samples may be added directly to scintillation vials containing scintillant, capped and inverted to mix Count the samples using an appropriate program. Note: If no scintillant was added, 32 P-labeled samples may be counted using a 1 minute Cerenkov counting program. Samples mixed with scintillant may be counted using a standard 32 P counting program Add the cpm obtained for each set of duplicate samples and divide by two to obtain the average. 16

17 Percent label incorporation may be calculated using the formula: % label incorporation = average cpm for COLUMN sample X 100 average cpm for TOTAL sample Note: Percent incorporation values obtained using this product may vary from values previously obtained by standard TCA analysis or ethanol precipitation. This product is not designed to directly replace these methods but is meant to offer a fast (4 minutes/analysis) alternative to standard techniques. 17

18 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. Possible cause Things to note when using ProbeQuant G- 50 spin columns. Suggestions 1. After the pre-spin (to remove the STE buffer in the columns) the resin will appear dry, and may pull away from the sides of the column. 2. Ensure the pre-spun column is used right away - it should not be allowed to sit very long or the column can get too dried and cracked. If this happens, the sample can run right through the cracks. Also, if the column is allowed to over-dry, DNA can non-specifically bind to the Sephadex. 3. CAREFULLY pipette the sample onto the top of the resin bed, and make sure the solution does not run into the sides of the resin bed. 4. The sample volume should be 50 μl (if less than 50 μl, bring volume up to 50 μl prior to loading). If sample volume is greater than 50 μl, unincorporated labeled nucleotides may elute off. Therefore, 18

19 Possible cause Suggestions perform multiple purifications using separate ProbeQuant columns. 5. After the spin, the elution volume should be ~50 μl. If it is more than 50 μl, either the column was not initially pre-spun correctly, or the spin time for the elution step was too long. If the sample volume is much less than 50 μl, check spin time and speed. Possible cause Columns were dried and cracked after the first centrifugation step. Suggestions 1. Cracking of the column bed in such a way is usually indicative of overcentrifuging and subsequent drying out of the column packing. Ensure that columns are spun at the correct speed and for the appropriate time. If the centrifuge is correctly calibrated, then add 250 μl of Probe Elution and Storage Buffer to the column and recentrifuge using the correct settings. 2. Do not use the pulse button on the microcentrifuge as it could override the speed setting, and inadvertently reset centrifuge to the top speed. 3. Ensure timing of the spins are correct, i.e., 1 minutes prespin and 2 minutes elution spin. 4. If the column is still considered to be too dry, reduce speed of centrifuge by 20% 19

20 Possible cause When spinning out the void volume before loading on sample, the resin comes away from the sides of the column. Suggestions 1. The resin does come away slightly to form a column with in the tube. It is essential that the sample being purified is added slowly onto the top of the resin as shown in the diagram in section

21 6.2. Related products available from GE Healthcare Product Number RPN6101M Product Blotting Hybond blotting paper Pack sizes 100 Sheets AA0085 RPN RPN1604 RPN1606 RPN1509 N5500 N5000 RPN1635 Labeling Redivue nucleotides Rediprime II DNA labeling system Ready-To-Go DNA Labeling Beads (-dctp) Megaprime DNA Labeling System, dntp Megaprime DNA Labeling System, dctp 5 -End Labeling Kit NICK Translation Kit, dntp NICK Translation Kit, dctp Rapid-hyb Buffer 250UCI - 1MCI 30 reactions 1 kit 30 reactions 30 reactions 20 reactions 20 reactions 20 reactions 125 ml RPN6K RPN6L US Detection Hyperfilm MP Hyperfilm MP Enveloped Purification NICK columns MicroSpin G-50 columns MicroSpin G-25 columns ExoSAP-IT NAP -5 columns GFX PCR DNA and Gel Band Purification kit GFX 96 PCR purification kit 25 sheets 50 sheets 50 columns 50 columns 50 columns 500 rxns 50 columns 100 columns 10 x 96 well plates 21

22 Product Number Product AutoSeq G-50 CyScribe GFX purification kit Amplification Taq DNA Polymerase See GE Healthcare catalogue Ready-To-Go PCR Beads (100 reactions in 0.5 ml tubes) DNA Polymerization Mix Pack sizes 50 columns 50 purifications A full range of sequencing kits and reagents can be found in the GE Healthcare catalogue. 22

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24 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: Norway Tel: Fax: Portugal Tel: Fax: Russia, C.I.S. & N.I.S Tel: Fax: GE Healthcare UK Limited Amersham Place Little Chalfont Austria Tel: 01/ Fax: 01/ Italy Tel: Fax: Spain Tel: Fax: Buckinghamshire HP7 9NA UK Belgium Tel: Fax: Japan Tel: Fax: Sweden 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, & South East Europe Tel: Fax: Denmark Tel: Fax: Eire Tel: Korea Tel: Fax: Latin America Tel: Fax: Middle East & Africa Tel: Fax: Netherlands Tel: Fax: Switzerland Tel: Fax: UK Tel: Fax: USA Tel: Fax: Fax: Finland & Baltics Tel: Fax: GE Healthcare UK Limited Amersham Place, Little Chalfont Buckinghamshire, HP7 9NA UK imagination at work PL Rev A 2006

25 The next four 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.

26 illustra Product protocol card ProbeQuant G-50 Micro Columns, Radiolabeled Probe Purification Kit (For the removal of unincorporated labeled nucleotides from DNA labeling reactions) 1. Perform the radiolabeling reaction. 2. Adjust the reaction volume to 50 μl with Probe Elution and Storage Buffer. 3. Vortex to resuspend resin. 4. Loosen cap and snap off the bottom closure. 5. Place the column in a collection tube (supplied). Experienced user protocol 6. Pre-spin. 1 minute at 735 x g PC Rev A 2006

27 ProbeQuant G-50 Micro Columns, Radiolabeled Probe Purification Kit 7. Insert column in 1.5 ml microcetrifuge tube. Slowly add sample to the top-center of the resin. 2 minutes at 735 x g 8. Elute sample. GE and GE monogram are trademarks of General Electric Company. ProbeQuant and illustra are trademarks of GE Healthcare companies 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 Representative for the most current information and a copy of the terms and conditions imagination at work GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK