Purification of the sequencing reaction product prior to electrophoresis is essential to remove excess, unbound dye terminators. Centrifugation columns can be a fast and efficient way to remove unbound dye terminators. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

Purification of sequencing reaction products

Materials and Instruments

Formamide Nucleic acids Oligonucleotides
ABI PRISM 3100Genetic Analyzer Microcentrifuge Tubes DTR Gel Filtration Cartridges (Edge Biosystems) or appropriate vacuum concentrator

Move

I. Materials

1. Buffers and solutions

Sample Sampling Solution

The sample preparation used depends on the equipment used. For the ABI PRISM 3100 Genetic Analyzer, it is recommended to use a highly detachable substrate.

formamide (e.g. ABI Hi-Di-Formamide).

2. Nucleic acids and oligonucleotides

The sequencing reaction product obtained in Scheme 3, II, Methods, Step 5

3. Special equipment

ABI PRISM 3100 Genetic Analyzer, microcentrifuge tubes, DTR Gel Filtration Cartridges (Edge Biosystems) or corresponding vacuum concentrator

II. Methods

1. Remove the required number of assembled cylinders from the bag and carefully inspect them to ensure that the plates in the cylinders are not dry. If there is no excess liquid in the tubes or the plates are dry, add 200 ul of deionized water and allow the plates to rehydrate for a few minutes.

2. Place the cylinder in a microtube and centrifuge at 1360g for 2 minutes (e.g. 4000r/min in an Eppendorf 5415C with a fixed inclination rotor). This step is necessary, otherwise excess water will be added to the cylinders.

3. Remove the cylinder and place it into another clean tube and carefully add the sample to the middle of the plate mold.

4. Place the cylinder back into the centrifuge and keep it in the same direction as the first centrifugation and centrifuge at 1360g for 1~1.5min.

Shorter centrifugation times result in lower recoveries, typically 75%-85%, but almost complete removal of unbound dye. Longer centrifugation times may increase the recovery (95%), but the eluate will contain termination dye, resulting in ambiguous bases near the primer.

5. The eluate can be frozen at -20°C or analyzed immediately by automated sequencers.

6. Concentrate the eluate in its empty concentrator for 20~30min without heating, because the dye is light sensitive, the concentration time should not be too long, otherwise it may lead to degradation.

7. Resuspend the sample with 10ul of sampling buffer according to the manual of the sequencer, the sampling director depends on the sequencer used, for ABI PRISM 3100 Genetic Analyzer, 1~l.5ul per capillary can be added.

8. Follow the instructions of the sequencer for electrophoretic separation and sample analysis.

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