p47

cloning . . .

Growth

  1. Transform BL21 DE3 [RIL] with DNA. Select on Amp plates.
  2. Pick a single colony into 1 l 2xTY/Amp. Grow overnight at 37°C with shaking.
  3. Harvest by centrifugation at 5,000 rpm at 4°C for 10 minutes.
  4. Resuspend in 10 ml sucrose buffer per litre of media. Store at -20°C.

Purification

  1. Thaw pellets and lyse by sonication on ice (4 x 20s on 25% power with the medium tip should do it. Don't overcook).
  2. <20k, 2°C, 20 min>
  3. Dialyse at 4°C overnight versus 4l buffer.
  4. <20k, 2°C, 20 min>
  5. Load on to -Sepharose in buffer. Keep the flow-through. Elute with a 100 ml gradient to .
  6. Concentrate the -containing fractions in Centriprep-10 (or Vivaspin 10) and apply to S-100 in buffer + 100 mM NaCl.
  7. Collect TapC2-containing fractions and concentrate (Centriprep/con 3/V5).

Quantification

Coefficient at 280 nm = 10,360 M-1 cm-1.

Mr = 43,157.

Enter
A280 reading 		Enter
dilution factor 		  Conc (mM) Conc (mg ml-1)

Buffers

HiLo HiHi
20 mM Tris-Cl pH 8.5
1 mM EGTA
1 mM DTT
 		 20 mM Tris-Cl pH 8.5
1 mM EGTA
1 mM DTT
500 mM NaCl
RPG 2000/07/03

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