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Protocol for Generation of Unidirectional Deletions in DNA Fragments
(for 25 time points, with Exonuclease III, E.coli)

1. Digest 5-10 µg of DNA with a pair of restriction enzymes; one which generates a blunt or 5'-overhanging end adjacent to the target sequence, and another which produces a resistant 4 base 3'-overhang close to the priming site.
2.  
   1. Extract reaction mixture with:
      • 1 volume of phenol:chloroform (1:1)
      • then with 1 volume of chloroform:isoamyl alcohol (24:1).
   2. Precipitate DNA by adding:
      • 0.1 volume of NaCl/glycogen solution (1.1M NaCl, 0.25 mg/ml glycogen)
      • 2 volumes of 95% ethanol.
   3. Mix and centrifuge at 10,000 rpm for 10 minutes.
   4. Wash the pellet with 1 ml of 75% ethanol.
   5. Dry the pellet.
3. Dissolve DNA in 50 µl of 1X Exonuclease III reaction buffer.
4. Prepare mixture (S1 Nuclease mix) by adding the following:
   

   5X reaction buffer for S1 Nuclease	40 µl
   S1 Nuclease	0.5 µl (50 u)
   Water, nuclease-free	to 200 µl

5. Add 7.5 µl aliquotes of the S1 Nuclease mix into each of 25 numbered tubes, place on ice.
6. Select incubation temperature and time as follows:

   Temperature, °C	25	30	37	45
   Digestion rate, bp/min	80	210	420	600

   Mononucleotides are released at base-dependent rates in the order:
   C>A=T>G. Different termini of DNA are degraded at different rates, so these guidelines should only be used as an approximation.

7. Warm the DNA solution to the digestion temperature. Add 500 units of Exonuclease III and mix rapidly. Remove 2 µl aliquots at the chosen time intervals and add to the tubes containing the cold S1 Nuclease mix.
8. After all samples have been taken, move the tubes to room temperature for 30 minutes. 
   Add 1 µl of S1 "STOP" solution (300 mM Tris base, 50 mM EDTA) and heat at 70°C for 10 minutes to inactivate S1 Nuclease.
9. Use 7 µl of each sample for gel electrophoresis.

Reference

1. Henikoff, S., Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing, Gene, 28, 351-359, 1984.

Ordering Information

Exonuclease III S1 Nuclease Nb.Bpu10I Glycogen Water, nuclease-free

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Updated gegužės 09, 2007 16:40