Quick-StartProtocol DyeEx® 2.0 Spin Kit January 2011

Quick-StartProtocol DyeEx® 2.0 Spin Kit January 2011
Quick-StartProtocol
DyeEx® 2.0 Spin Kit
The DyeEx 2.0 Spin Kit (cat. nos. 63204 and 63206) can be stored at room
temperature (15–25°C) for up to 12 months.
For more information, please refer to the DyeEx Handbook, which can be found
at www.qiagen.com/handbooks.
For technical assistance, please call toll-free 00800-22-44-6000, or find
regional phone numbers at www.qiagen.com/contact.
Notes before starting
„
„
This protocol is suitable for sequencing reactions with volumes of 10–20 μl.
For more reproducible pipetting and reduced error with sample volumes
<10 μl, we recommend adjusting the volume to 20 μl using distilled water
before application to the gel bed.
All centrifugation steps are carried out at 750 x g in a conventional
microcentrifuge at room temperature (15–25°C). The appropriate speed for
individual centrifuges can be calculated as follows:
rpm = 1000 x √ 750/1.12 r (r = radius of rotor in mm).
1.
Gently vortex the spin column to resuspend the resin.
2.
Loosen the cap of the column a quarter turn to avoid a vacuum inside the
spin column.
3.
Snap off the bottom closure of the spin column, and place the spin column
in a 2 ml collection tube (provided).
4.
Centrifuge for 3 min at the calculated speed.
January 2011
Sample & Assay Technologies
Quick-StartProtocol
5.
Carefully transfer the spin column to a clean centrifuge tube. Slowly apply
the sequencing reaction directly onto the center of slanted gel bed surface.
Note: Do not allow the reaction mixture or the pipet tip to touch the sides
of the column. The sample should be pipetted slowly so that the drops are
absorbed into the gel and do not flow down the sides of the gel bed. Avoid
touching the gel bed surface with the pipet tip.
Note: It is not necessary to remove mineral oil or kerosene prior to cleanup
of dye-terminator sequencing reactions.
Note: It is not necessary to replace the lid on the column.
6.
Centrifuge for 3 min at the calculated speed. Remove the spin column from
the microcentrifuge tube. The eluate contains the purified DNA. For most
sequencers, it is possible to load the eluate directly onto the sequencer.
Optional: If using a formamide loading buffer, dry the samples in a
vacuum centrifuge and proceed according to the instructions provided with
the DNA sequencer.
For up-to-date licensing information and productspecific disclaimers, see the respective QIAGEN® kit
handbook or user manual.
Trademarks: QIAGEN®, DyeEx® (QIAGEN Group). 1066978 01/2011
© 2011 QIAGEN, all rights reserved.
Sample & Assay Technologies
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