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Home :: Technical Resources :: Sequence Reaction Cleanup
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Sequence Reaction Cleanup
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Before loading samples on a sequencing instrument, unincorporated dye terminators and primers need to be removed. This will avoid much of the background noise and dye peaks in chromatograms. There are many methods available to clean sequence reactions. The method outlined below uses shrimp alkaline phosphatase (SAP), 1-butanol, and a Sephadex™ G-50 matrix in a stepwise fashion.
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Preparation of Sephadex™ filter plates:
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Sephadex™ matrix should be hydrated before starting this procedure. Plates can be prepared by the method described below.
- Fill wells of a 45 µl 96-well multiscreen column loader (Millipore) with Sephadex™, then invert it into a 96-well filter plate.
- Add 300 µl of sterile distilled water into each well.
- Wrap plate tightly in plastic and keep at room temperature for 3 hours.
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Extra filter plates can be prepared and stored. After the 3 hour room temperature incubation they can be stored at 4°C for up to 2 weeks. They should be wrapped as tightly as possible to ensure that they stay hydrated.
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Addition of SAP:
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It is recommended to use 1 unit of SAP for each sample. If the concentration of SAP is at 1 U/µl, it is recommended to prepare a 1:2 dilution and distribute 2 µl to each sample. Distributing a mix will result in a loss of volume, so that additional mix is needed for a fixed number of reactions. For example, for 48 reactions make enough mix for 52.
- Calculate the volume of SAP needed for the number of samples being treated (52 µl).
- Prepare a 1:2 dilution of this amount of SAP with sterile distilled water (52 µl of SAP in 52 µl of water).
- Add 2 µl of the dilution to each sample.
- Centrifuge briefly, then incubate at 37°C for 30 minutes.
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Addition of 1-butanol:
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The 10% 1-butanol should be freshly prepared.
- Calculate the volume of 10% 1-butanol needed for the number of samples being treated (520 µl).
- Prepare the 10% 1-butanol with sterile distilled water (70 µl of 1-butanol in 630 µl of water).
- Add 10 µl of the 10% 1-butanol to each sample.
- Centrifuge briefly, then immediately proceed to the Sephadex™ cleanup.
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Filtering through Sephadex™ G-50 matrix:
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First make a visual check to see that the wells in the prepared filter plates are still hydrated. Do not use plates that are dry.
- Place a 96-well microplate under the filter plate.
- Centrifuge at 900 X g for 5 minutes, then discard the water.
- Place an optical 96-well reaction plate under the filter plate.
- Add prepared samples (samples + SAP + 1-butanol) into the filter plate.
- Centrifuge at 900 X g for 5 minutes.
- The samples can now be dried at 70°C.
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