PRINCETON SEPARATIONS Centri-Spin 10 Columns Kit Instructions

PRINCETON SEPARATIONS Centri-Spin 10 Columns Kit

Product Information

Specifications:

• Product Name: Column Hydration Kit
• Model Number: CH-100
• Capacity: 1 spin column
• Recommended Applications: DNA purification, nucleic acid isolation

Product Usage Instructions

1. Column Hydration
2. Gently tap the column to ensure the dry gel has settled at the bottom.
3. Remove the top column cap and reconstitute the column by adding 0.65 ml of reagent grade water or buffer of choice. Replace the column cap and vortex vigorously for about 5 seconds. Remove air bubbles by tapping the bottom of the column.
4. Removal of Interstitial Fluid
5.  After hydrating the gel and removing bubbles, remove the top column cap and then the column end stopper from the bottom.
6.  Spin the column and wash tube in a variable-speed centrifuge at 750 x g for 2 minutes to remove interstitial fluid. Keep track of the column position using the orientation mark if using a fixed-angle microcentrifuge.
7. If there is a drop at the end of the column, blot it dry. Discard the wash tube and interstitial fluid. Process the sample promptly to avoid excessive drying of the gel material.

FAQ

1. Q: Can I reuse the spin column after a single use?
   A: It is recommended to use a new spin column for each sample to ensure optimal results and prevent cross-contamination.

2. Q: What is the maximum recommended spin speed for this column?
   A: The recommended maximum spin speed is 750 x g. Exceeding this speed may damage the column and affect results.

CENTRI SPIN -10 COLUMNS For Research Use Only

PRINCIPLE
CENTRI SPIN -10 Columns are used for the fast and efficient purification of large molecules (peptides, proteins, nucleic acids, complex carbohydrates) from small molecules (nucleotides, labels and buffer salts). The column design is based on the description by Sambrook, et al. (1) of gel filtration for the purification of DNA from nick translation reactions. Each unit consists of a special fritted microfuge tube, dry gel, wash tube and sample collection tube.

• The gel will provide excellent recovery of DNA fragments >10-mer or 10 base pairs while removing >98% of salts, NTP’s and other low- molecular-weight compounds.
• The column gel is hydrated with reagent- grade water or a suitable buffer and spun in a microcentrifuge or swinging-bucket centrifuge to remove the interstitial fluid. The sample is then applied and the column is spun again, processing the sample. The sample is purified by the retention of low-molecular-weight contaminants in the matrix, while the larger molecules of interest are exchanged into the buffer of choice and eluted into the collection tube.
• These columns are far superior — in ease of use, speed, and non-toxicity – to such common techniques as phenol/chloroform extraction, ethanol precipitation, dialysis and ultrafiltration.

Benefits include

• RAPID AND EFFICIENT SEPARATIONS
• BUFFER NOT PRESELECTED
• COLUMNS STABLE AT ROOM TEMPERATURE
• CONVENIENT 20-50l SAMPLE SIZE

CENTRIFUGE NOTES

• Maximum yield and efficiency are obtained with the horizontal or swinging-bucket rotors. However, fixed-angle-rotor microcentrifuges provide acceptable performance and save time.
• On a variable speed microcentrifuge, DO NOT use the pulse button, which overrides the speed setting and takes the rotor to maximum g-force. If you are not sure of the g-force generated by your centrifuge at specific speeds, calculate the correct speed by using the following formula:
• Where rpm = revolutions per minute;
• RCF = Relative Centrifugal Force and
• r = radius (cm) measured from center of spindle to bottom of rotor bucket.
• Example: For RCF = 750 and r = 7.5 cm

QUALITY CONTROL:

• Every batch of CENTRI
• SPIN-10 Columns is tested for separation efficiency and fill accuracy.

MATERIALS PROVIDED

• CENTRI
• SPIN-10 Columns containing dry gel
• Wash Tubes (2 ml) • Sample Collection Tubes (1.5 ml)

ADDITIONAL MATERIALS RECOMMENDED

• Microcentrifuge (Eppendorf 5415C, Variable Speed or equivalent)
• Variable pipets (Pipetman 100 ml)
• Pipet Tips
• Pipet Bulb, Dispo, 2ml Latex
• Microtube Rack
• Vortex Mixer

COMMON PROBLEMS

1. A failure to remove excess interstitial fluid after hydration of the columns.
2. Touching the side of the column during sample application. Both errors can result in ineffective separation.

SOLUTIONS

1. Note if any columns have released less fluid than the others during the first spin. Simply spinning them again briefly will usually remove the excess fluid.
2. Load the sample directly into the center of the gel bed and do not touch the sample to the walls of the column.

REFERENCE
Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory, 1989.

CENTRI • SPIN-10 Protocol

CENTRI • SPIN-10 Columns have been designed specifically for the following uses:

• Purification of primers or probes ≥ 10 bases
• Purification of biotinylating reactions
• Purification of protein conjugates in
• iodination
• fluorescence labeling
• cross-linking
• haptenation
• Desalting/purification/buffer exchange of pep- tides or proteins prior to electrophoresis

CENTRI • SPIN-10 is designed For Research Use Only

The following protocol may be used for all recommended applications.

1. COLUMN HYDRATION

2. Gently tap the column to insure that the dry gel has settled in the bottom of the spin column.

3. Remove the top column cap and reconstitute the column by adding 0.65 ml of reagent grade water or buffer of choice. Replace the column cap and vortex vigorously for ~ 5 seconds. Remove air bubbles by sharply tapping the bottom of the column. It is important to hydrate all of the dry gel.

4. Allow at least 30 minutes of room temperature hydration time before using the columns. Reconstituted columns may be stored refrigerated at 4°C for several days. Longer storage can be accomplished in 10 mM sodium aside (NaN3). Allow refrigerated columns to warm to room temperature before use.

5. REMOVAL OF INTERSTITIAL FLUID

6. After the gel has hydrated and is free of bubbles, first remove the top column cap, and then remove the column end stopper from the bottom.

7. Spin the column and wash tube in a variable speed centrifuge at 750 x g for 2 minutes to remove interstitial fluid. (For example, for Eppendorf Model 5415C, spin at 3000 rpm for 2 minutes.) If you use a fixed-angle microcentrifuge, keep track of the position of the column using the orientation mark molded into the column.

8. If there is a drop at the end of the column, blot it dry. Discard the wash tube and the interstitial fluid. Do not allow the gel material to dry excessively. Process the sample within the next few minutes.
   EXPERIMENTAL RESULTS

9. SAMPLE PROCESSING

10. Hold the column up to the light. Transfer 20 to 50 Ul of the sample to the top of the gel. Carefully dispense the sample DIRECTLY ONTO THE CENTER OF THE GEL BED at the top of the column, without disturbing the gel surface (See Figure 1). DO NOT contact the sides of the column with the reaction mixture or the sample pipet tip, since this can reduce the efficiency of purification.

11. Place the column into the SAMPLE COL- LECTION TUBE (1.5 ml) and place both into the rotor. Maintain proper column orientation. The highest point of the gel media in the column should always point toward the outside of the rotor. (See Figure 1). Spin the column and collection tube at 750 x g for 2 minutes. The purified sample will collect in the bottom of the Sample Collection Tube. Discard the spin column and continue with your procedure.

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