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, 19-Jun-2014 11:59:50 PDT
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Eric Kofoid eckofoid at ucdavis.edu