Simplified preparation of unidirectional deletion clones

The unidirectional digestion of DNA, first cut with two different restriction enzymes generating one 3'-protrusion and than with exonuclease III and Sl-nuclease allows a very defined removal of DNA for sequencing (1,2) or mapping of biological functions. The DNA is cloned into the polylinker of e.g. pUC18 or mpl8 (2), double digested with SstI/SmaI or PstI/XbaI, phenol extracted, ethanol precipitated and taken up in 10 mM Tris-HC1, 0.1 mM EDTA pH 8.0 at 0.2 mg/ml. For avoiding further phenol and ethanol steps (1) the following protocol is very helpful: 12.5 41 linearized DNA, 5 41 5x-Exo-buffer (75 mM Tris-HCl pH 8.0, 3.3 mM MgCi2) and water to final 25 4 are mixed and incubated for 2 min at the desired temperature (200 base pairs/min: 370C, 30/min: 22°C), than 135 units exonucleaseIII/pmole susceptible 3' ends are added at time zero (about 190 units for 2.5 4g pUC18). Every minute a 3 41 aliquot is mixed quickly with 3 4 water, which is heated to 1000C, and incubated for 5-7 minutes at 700C to stop and inactivate exonuclease III. On ice 15 l Si-buffer (16 mM Na-acetate pH 4.6, 400 mM NaCl, 1.6 mM ZnSO4, 8% glycerol) and 4 l Sl-nuclease (4.5 U/4) are added and incubated at room temperature for 10-20 min to digest single stranded DNA and stopped by a pH-shift upon adding 5 41 of 800 mM Tris-HCl pH 8.0, 20 mM ETDA pH 8.0, 80 mM MgCl2. (8 41 are run on a 1% agarose gel to check the extend of deletion). The remaining 22 41 are incubated for 2 min at 37°C with 2 41 of Klenowfragment; 2 4 of a mixture of the four dXTP's (each at 0.25 mM) are added for 10 min at 370C to create blunt ends. 13 41 ligation-buffer (80 mM Tris-HCl pH 7.5, 30mM DTT, 20 mM MgC12, 3mM spermidine), 2 41 of 10 mM ATP and 4 41 T4-DNA ligase are added and the ligation run for 6 hrs at 25°C or overnight at 18°C. Calcium treated competent JM83 cells are transformed with 10 41 of the ligation mix and spread on LB-plates with ampillicin (100 4g/ml). About thousand colonies (1-2% of that for ccc-DNA) are obtained. 1. Henikoff, S. (1984) Gene 28,351-359. 2. Yanisch-Perron,C., Vieira,J. and Messing,J. (1985) Gene 33,103-119.