-
Prepare 200 μl of 10 mM Tris-HCl pH 7.5 for later use.
-
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 |
-
Transfer 3.5 ml of 2X wash/bind buffer (5 mM Tris-HCl pH 7.5, 1 M NaCl, 0.5 mM EDTA) to a clean 15 ml Falcon tube.
-
Add 3.5 ml of nuclease-free water to the same 15 ml Falcon tube to make 7 ml of 1X wash/bind buffer.
-
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.
-
Spin down the tube and pellet the beads on a magnet for two minutes, then pipette off the supernatant.
-
Repeat steps 7 and 8 two more times for a total of three washes.
-
Important
It is critical that 2X buffer is used for the next step. Using 1X buffer will result in inefficient binding.
-
Resuspend the beads in 10 μl of 2X wash/bind buffer to achieve a final bead concentration of 5 μg/μl.
-
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.
-
Important
In the next steps, it is critical to pellet the beads on the magnet for the specified timings to ensure none are left in solution as the beads are difficult to see.
-
Add 1 ml of 1X wash/bind buffer and vortex the DNA and beads with buffer for 5 seconds.
-
Spin down the tube and pellet the beads on a magnet for three minutes, then pipette off the supernatant. Take care to not aspirate any of the beads.
-
Repeat the steps 13 and 14 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.
-
End of step
Take forwards 20 μl of the amplicon-bead conjugate into the post-pull-down PCR step.