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Prepare 4 ml of 2X wash/bind buffer (10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA).
Reagent |
Stock concentration |
Final concentration |
Volume |
Tris-HCl pH 7.5 |
1 M |
10 mM |
40 μl |
NaCl |
5 M |
2 M |
1600 μl |
EDTA |
0.5 M |
1 mM |
8 μl |
Nuclease-free water |
- |
- |
2352 μl |
Total |
- |
- |
4000 μl |
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Transfer 3.5 ml of the 2X wash/bind buffer to a fresh 15 ml Falcon tube and add 3.5 ml of nuclease-free water to make 7 ml of 1X wash/bind buffer (5 mM Tris-HCl pH 7.5, 1 M NaCl, 0.5 mM EDTA).
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Resuspend the M280 streptavidin beads (10 μg/μl) by vortexing.
-
Transfer 5 μl of the streptavidin beads to a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Add 1 ml of 1X wash/bind buffer and vortex the beads with buffer for 5 seconds. Pellet the beads on a magnet for two minutes, then pipette off the supernatant.
-
Repeat the previous step two more times for a total of three washes.
-
Resuspend the beads in 10 μl of 2X wash/bind buffer to achieve a final bead concentration of 5 μg/μl.
-
Important
It is critical that 2X buffer is used for this step. Using 1X buffer will result in inefficient binding.
-
Add 10 μl of 5 μg/μl prepared beads (50 μg beads total) to the tube with 10 μl of biotinylated cDNA.
-
Incubate on a Hula mixer (rotator mixer) for 20 minutes at room temperature.
-
Add 1 ml of 1X wash/bind buffer and vortex the DNA and beads with buffer for 5 seconds. Pellet the beads on a magnet for two minutes, then pipette off the supernatant. Take care to not aspirate any of the beads.
-
Repeat the previous step two more times for a total of three washes.
-
Add 200 μl of 10 mM Tris-HCl pH 7.5 and vortex the beads for 5 seconds.
-
Spin down and place the tube back on the magnet for three minutes. Pipette off the supernatant.
-
Remove the tube from the magnetic rack and resuspend the pellet in 20 μl of nuclease-free water. Vortex for 5 seconds and briefly spin down to collect the amplicon-bead conjugate.