G-25 Spin Column provides a fast and efficient purification of large molecules (nucleic acids, complex carbohydrates, peptides, proteins) from small molecules (nucleotides, labels and salts). Each column contains a special frit and pre packaged with wet or dry resin designed to achieve high recovery (>70%) of DNA fragments (>10 bases) while removing >98% of salt, traces of phenol, probes, and dNTP’s. 


--Idea for the purification of oligonucleotides or very small DNA fragments (>10 bases). 

--Fast & efficient removal of free and labeled dNTPs in end-labeling, nick translation, and other synthesis or labeling reactions. 

--Removal of dye deoxyterrminators in manual or automated sequencing reactions. 

--Buffer exchange, purification of peptides or proteins. 

--Commonly used for purification of protein conjugates in fluorescence, cross-linking, or other labeling reactions. 

Protocol for G25W (wet gel): 

Note: Maximum yield and efficiency are obtained with horizontal or swinging-bucket type rotors. However, microcentrifuges with fixed-angle-rotor provided acceptable performance. Do not use pulse button on a variable speed microcentraifuge, which overrides the speed setting to maximum speed. 

1. Shake the bottle containing Sephadex G25 resin to make sure the gel slurry is fully resuspended and use 1ml pipette to transfer 0.75ml resin into each ESC7 column. 

2. Spin at low speed (750x g) for 20 seconds to remove the storage buffer (ddH2O+10mM Sodium Azide). 

3. Equilibrate the column by adding 350l of the buffer of your choice (or ddH2O) to the top of the resin and spin at 750xg for 20seconds. Discard the flow-through. Repeat this step twice. 

4. Make sure the gel is free of bubbles. If there is a drop at the end of the column, blot it dry. 

Note 1: If using a fixed-angle microcentrifuge, keep track of the orientation of the column in the rotor. The highest point of the gel media in the column should always point toward the outside of the rotor. 

Note 2: Make sure columns do not crack or dry after spin, if so, resuspend the resin again and centrifuge for less time or up to 20% slower speed. Columns do not operate efficiently if they have dried out. 

5. Immediately apply20-50ul of sample to the center of the gel bed at the top of the column carefully without disturbing the gel surface. 

Note 1: To obtain highest purification efficiency, do not contact the sides of the column with sample pipette tip or reaction mixture. Note 2: Sample volume should not exceed 100ul. 

6. Place the column with a new sample collection tube (2.0 ml) into the rotor. 

Note: It is important to maintain proper column orientation. The highest point of the gel media in the column should always point toward the outside of the rotor. 

7. Spin the column with collection tube at 4oC for 2 minutes at 750x g and your sample will be the flow-through in the collection tube. Discard the spin column and continue with your procedure 

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