Plasmids and methods for monitoring endonuclease digestion efficiency

A method for designing a plasmid for use in monitoring the efficiency of a restriction endonuclease digestion comprising:

• (a) identifying at leas t one spacer segment comprising a nucleic acid sequence that is restriction site-free;
• (b) identifying a plurality of restriction sites to be represented o n said plasmid;
• (c) assigning each of said restriction sites to a polylinker region on said plasmid such that for any two restriction sites, the two sites are situated in different polylinkers;
• (d) distributing said polylinker regions on said plasmid such that said polylinker regions are separated by a spacer segment at least about 15%-85% of the length of the plasmid.

A method for designing a set of plasmids for use in monitoring the efficiency of a restriction endonuclease digestion comprising:

• (a) identifying at least one spacer segment comprising a nucleic acid sequence that is restriction site-free;
• (b) identifying a plurality of restriction sites to be represented on said plasmids;
• (c) assigning each of said restriction sites to a polylinker region on one of said plasmids such that for any two restriction sites, there is at least one plasmid in the set in which the two sites are situated in different polylinkers;
• (d) distributing said polylinker regions on said plasmids such that said polylinker regions are separated by a spacer segment at least about 15%-85% of the length of the plasmid.

A method for designing a set of plasmids for monitoring the efficiency of a restriction endonuclease digestion comprising:

• (a) identifying at least one spacer segment comprising a nucleic acid sequence that is restriction site-free;
• (b) identifying a plurality of restriction sites to be represented on said plasmids;
• (c) determining the number of polylinker regions (a) that will accommodate said restriction sites, wherein the maximum number (N) of sites which can be represented is N=ab, where a is the number of polylinkers in each plasmid, b is the number of plasmids in the set;
• and
• (e) assigning each of said restriction sites to a polylinker region in accordance with a template, wherein said template corresponds to an a×b matrix, and wherein each of said sites is in a different polylinker from any of the other sites in at least one of the plasmids in the set.

A method for designing a set of three plasmids for monitoring the efficiency of a restriction endonuclease digestion comprising:

• (a) identifying at least one spacer segment comprising a nucleic acid sequence that is restriction site-free;
• (b) identifying 27 restriction sites to be represented on said plasmids;
• (c) numerically ordering said restriction sites;
• (d) assigning each of said restriction sites to a polylinker region, wherein
• (i) sites 1-9 are assigned to a first polylinker on a first plasmid, sites 10-18 are assigned to a second polylinker on said first plasmid and sites 19-27 are assigned to a third polylinker on said first plasmid;
• (ii) sites 1, 4, 7, 10, 13, 16, 19, 22 and 25 are assigned to a first polylinker on a second plasmid, sites 2, 5, 8, 11, 14, 17, 20, 23, and 26 are assigned to a second polylinker on said second plasmid and sites 3, 6, 9, 12, 15, 18, 21, 24, and 27 are assigned to a third polylinker on said second plasmid;
• (iii) sites 1, 2, 3, 10, 11, 12, 19, 20 and 21 are assigned to a first polylinker on a third plasmid, sites 4, 5, 6, 13, 14, 15, 22, 23 and 24 are assigned to a second polylinker on said third plasmid and sites 7, 8, 9, 16, 17, 18, 25, 26 and 27 are assigned to a third polylinker on said third plasmid;
• and
• (e) distributing said polylinker regions on each of said plasmids such that said polylinker regions are separated by a spacer segment at least about 15% of the length of the plasmid.

A method of constructing a set of four plasmids for monitoring the efficiency of a restriction endonuclease digestion comprising:

• (a) identifying at least one nucleic acid sequence that is restriction site-free;
• (b) identifying 64 restriction sites to be represented on said plasmids;
• (c) numerically ordering said restriction sites;
• (d) assigning each of said restriction sites to a polylinker region, wherein
• (i) sites 1-16 are assigned to a first polylinker on a first plasmid, sites 17-32 are assigned to a second polylinker on said first plasmid, sites 33-48 are assigned to a third polylinker on said first plasmid and sites 49-64 are assigned to a fourth polylinker on said first plasmid;
• (ii) sites 1-4, 29-32, 41-44 and 53-56 are assigned to a first polylinker on a second plasmid, sites 5-8, 17-20, 45-48 and 57-60 are assigned to a second polylinker on said second plasmid, sites 9-12, 21-24, 33-36 and 61-64 are assigned to a third polylinker on said second plasmid and sites 13-16, 25-28, 37-40 and 49-52 are assigned to a fourth polylinker on said second plasmid;
• (iii) every fourth site beginning with site number 1 is assigned to a first polylinker on a third plasmid, every fourth site beginning with site number 2 is assigned to a second polylinker on said third plasmid, every fourth site beginning with site number 3 is assigned to a third polylinker on said third plasmid;
• and every fourth site beginning with site number 4 is assigned to a fourth polylinker on said fourth plasmid.
• (e) distributing said polylinker regions on each of said plasmids such that said polylinker regions are separated by a restriction site-free region at least about 15% of the length of the plasmid.