DNA-agarose coupling reaction

DNA probes are made using one 5'biotinylated oligonucleotide and one unmodified oligonucleotide. This pair can be used in a PCR reaction for longer sequences, or directly annealed for shorter sequences.

Recover SA AGAROSE BEADS BY CENTRIFUGATION @ 2000 rpm, FOR 2 min!

• Take 200µl of streptavidin agarose (SA) (Sigma)
• Wash the SA beads twice in 1ml of water.
• Prepare the coupling reaction:
  200µl washed SA beads resuspended in water
  100µl biotinylated DNA (500ng/µl (~2pmol/µl) for ~400bp fragment or 200ng/µl (~5-6pmol/µl) for oligo)
  15µl 5M NaCl
  12.5µl 1M Na₂HPO₄ pH 6.9
  172.5µl H₂O
  500µl total volume
• Incubate the coupling reaction for 1 hour (or more) at RT with the end-over-end rotation.
• To check efficiency of the coupling, take 20ml of supernatant for 1% agarose gel, and 5µl of the SA beads. After electrophoresis the SA beads coupled with DNA stayed in wells, since the beads are to big to enter the gel.
• Wash the SA beads twice in 1ml of SCB (150mM NaCl, Na₂HPO₄ pH 6.9) and twice in 1ml 1xGSBB (gel shift binding buffer).
  

  5xGSBB 100mM Hepes pH 7,9 250mM KCl 25mM MgCl2 60% glycerol 2,5mM EDTA pH 8,0 0,5% Triton X-100

• Resuspend SA-DNA beads in 100µl of 1xGSBB so that the total volume is 200µl
• In 100µl of "dry" beads and 100µl of buffer the concentration of DNA is ~1 pmol/µl for ~400bp fragment or ~2.5-3 pmol/µl for oligos
• store at +4°C - do not freeze,

PROTEIN BINDING REACTION

• Prepare binding reaction:
  300µl embryo lysate (15 embryo equivalents) - cleared of yolk, etc.
  80µl 5xGSBB
  4µl 0,1M DTT (-20°C)
  20µl 10mg/ml Herring sperm DNA (Sigma #)
• Incubate for 10 min at RT with the end-over-end rotation.
• Add 50µL (for ~400bp fragment) or 20-30µL (for oligo) SA-DNA beads; use SA bead alone as control as well as SA-nontarget DNA as controls.
• incubate at RT for 20 min (no longer! as non-specific binding increases with longer incubations) with the end-over-end rotation.
• Spin the SA beads, and take 20µL of supernatant for an SDS gel.
• Wash the SA beads two times in 1ml of 1xGSBB. Be careful to do it gently, without vortrexing.
• Recover the beads by centrifugation and remove as much supernatant as possible without disturbing the beads.
• Resuspend the beads in 40µL 2xSDS loading buffer, and heat for 10 min at 90°C.
• Load on acrylamide SDS-PAGE gel.

OLIGO ANNEALING

50 µL 5’oligo (1mg/ml)
50 µL 3’oligo (1mg/ml)
50 µL 10xTEN
350 µL H2O

Use screw cap microtube, and boil the annealing reaction for 10 min in 1L beaker with plenty of water. Leave the annealing reaction to slowly cool down. You will need couple of hours or o/n for this.

Add 2 µL of glycogen (25 mg/ml, my shelf at –20°C) and 1ml 96% EtOH, mix and precipitate as usual.

Resuspend the pellet in 100 µL of H₂O, so the concentration of annealed oligo is 1 mg/µl.

10xTEN: 1M NaCl, 100 mM Tris, 10 mM EDTA pH 7.5