Generation of uni-directionalnesteddeletion sin plasmid DNA

1) Digest DNA with two restriction enzymes such that one end is ExoIII susceptible (blunt or 5' overhang) and the other is ExoIII resistant (5' recessed). The ExoIII susceptible end should be such that deletion will proceed in the direction desired.

2) Heat inactivate restriction enzymes at 85℃for 30 mins.

3) Phenol/chloroform, chloroform extract and alcohol precipitate digested DNA.

4) Redissolve DNA in 20μl 1x One Phor All buffer.

5) Add 20μl 2x ExoIII buffer and preincubate the mixture for at least 2 minutes at the desired temperature.

6) Prepare 31 sterile eppendorf tubes, each containing 3μl S1 nuclease/S1 nuclease buffer (20μl 5x S1 nuclease buffer, 79μl H2 O, 1μl S1 nuclease [50U/ul]), labelled as time (t)= 0-30 minutes. Place the tubes on ice.

7) Remove a 2μl aliquot of the pre-incubated deletion mix into the tube labelled t=0 and hold on ice. Return the deletion mix to the water bath at the correct temperature, add 1μl ExoIII (~100U), mix gently by pipetting up and down and return to the water bath.

8) Allow incubation to proceed and at the individual time points, remove 2μl of the deletion mixture to the corresponding S1 tube and incubate on ice.

9) After all time points have been teken, incubate all the S1 tubes simultaneously at room temperature for 30 minutes.

10) Add 1μl S1 Stop buffer to each sample and incubate at 65℃for 10 minutes.

Analysis of the deletion reaction products is achieved by loading half of each timed sample onto a neutral agarose gel and comparing band sizes to those of the original (t = 0) sample and those of molecular weight markers. If the rate of deletion is deemed to be too fast, NaCl, at concentrations ranging between 0-150mM, can be added to the exonuclease III buffer as this slows the rate of exonuclease digestion. Incubation of the deletion reaction at higher or lower temperatures also raises or lowers, respectively, the rate of deletion.

The remaining half of the reactions can be Klenow blunt ended, ligated together and introduced into E. coli .

S1 Nuclease Storage Buffer: 10mM Tris.Cl (pH 7.5), 50mM NaCl, 0.1mM Zn(OAc)2 , 50% (v/v) glycerol.

6x ExoIII Nuclease Buffer: (360mM Tris.Cl, pH 8.0, 4mM MgCl2 , 6mM DTT).

5 x S1 Nuclease Buffer: 150mM sodium acetate, pH4.6, 250mM NaCl, 5mM ZnCl2 , 5%(v/v) glycerol).

S1 Stop Buffer: 500mM Tris.Cl (pH8.0), 125mM EDTA (pH8.0).