Magnetic Strand Separation of DNA Strands
Eric Kofoid (27 June 1994)
Description
==========
This protocol contains instructions for purifying both strands of PCR-amplified DNA in a form
suitable for direct sequencing. Preparation of the biotinylated strand ("W-strand") is essentially
according to Ryk Ward's standard protocol using half as many DynaBeads, with no apparent
sacrifice in yield. Preparation of the "C-strand" is substantially different from the Dynal protocol,
and produces better product faster. Refer to the Dynal instructions for details on magnetic
separation.
Protocol
==========
- Wash beads
- vortex bead stock
- remove 10 無 beads to 20 無 HBWB in tube "Watson", vortex
- magnet, discard supernatant
- resuspend beads in 30 無 HBWB
- Bind DNA
- add 5 - 20 無 PCR product to washed beads, mix by flicking
- 15' x RT with occasional resuspension
- magnet, discard supernatant
- resuspend beads in 40 無 HBWB -- can be stored indefinitely at this point at 4。
- magnet, discard supernatant
- Denature DNA
- resuspend beads in 15 無 0.2 N NaOH
- 15' x RT with occasional resuspension
- magnet, add supernatant to tube "Crick", preloaded with 30 無 Solution III
- Further strand separation -- do following 3x
- resuspend beads in 50 無 0.2 N NaOH
- magnet, add supernatant to Crick tube
- resuspend beads in 40 無 HBWB
- magnet, discard supernatant
- resuspend beads in 50 無 TE
- magnet, discard supernatant
- W-strand
- resuspend beads in 25 無 H2O
- can be stored at -20。
- C-strand
- To Crick tube, add 1 無 glycogen solution and mix well
- Add 500 無 95% ethanol -- mix well
- 30' x ice
- 10' in microcentrifuge full on
- wash 1x with 400 無 70% ethanol
- draw off ethanol in vacuum jar without fully drying pellet
- resuspend in 25 無 H2O
- can be stored at -20。
5 無 is typically used in a Sequenase or Taquence reaction.
- Materials
- ==========
- DynaBeads or "Beads"
- DynaBeads M-280
- Dynal, Inc.
- 5 Delaware Drive
- Lake Success, NY 11042
- "Solution III" -- Potassium acetate, pH 4.8 (5 M acetate, 3 M K)
- 294 g KCH3CO2
- 115 ml HCH3CO2
- H2O to 1 liter (no need to check pH)
- HBWB -- High-salt "Binding & Wash Buffer"
- 10 mM Tris, pH 7.5
- 1 mM Na2EDTA
- 3 M NaCl
- TE
- 10 mM Tris, pH 7.4
- 1 mM Na2EDTA
- Glycogen -- 20 mg/ml; Boehringer Mannheim -- cat. 901-393
Last Update: Thursday June 19 2014
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Eric Kofoid
eckofoid at ucdavis.edu