Method of truncating both ends of a large piece of DNA

A method of truncating both ends of a DNA insert flanked by a wild type loxP site and a loxP511 site in a vector using loxP transposon plasmids comprising:

• a. Constructing a plurality of plasmids of identical sequence each containing a first transposon carrying a wild type loxP site;
• b. Transforming said plurality of first transposon containing plasmids into cells, wherein each containing a DNA insert in said vector carrying a first antibiotic resistance gene;
• c. Inducing each of said plurality of first transposons to insert into said DNA inserts in said cells;
• d. Infecting said cells containing said plurality of first transposons inserted into said DNA inserts with Phage P1, and wherein Cre protein is expressed by Phage P1 during said infection;
• e. Truncating first end portions of each of said DNA inserts, wherein each of said DNA inserts contains one of said inserted first transposons, and thereby creating a plurality of first end truncated DNA inserts with new first ends;
• f. Packaging said first end truncated DNA inserts into a P1 Phage head;
• g. Using said P1 Phage head to infect a first colony of fresh NS3516 cells to amplify said first end truncated DNA inserts by selecting on LB agar plates containing a first antibiotic whose resistance gene occurs in said vector, and thereby amplifying said plurality of first end truncated DNA inserts in a plurality of NS3516 cell colonies generated from said first colony of fresh NS3516 cells;
• h. Isolating said amplified plurality of different first end truncated DNA inserts in said vector to obtain a plurality of colonies, each colony with the same end truncation, from said first colony of fresh NS3516 cells;
• i. Using a first synthetic DNA primer to sequence said amplified plurality of new first end portions of said amplified plurality of different first end truncated DNA inserts;
• j. Constructing a plurality of plasmids of identical sequence each containing a second transposon carrying a loxP511 site and a second antibiotic resistance gene different from said first antibiotic resistance gene occurring in said vector;
• k. Transforming said plurality of second transposon containing plasmids into cells containing said amplified plurality of first end truncated DNA inserts;
• l. Inducing said plurality of second transposons to insert into said amplified plurality of first end truncated DNA inserts;
• m. Infecting said cells containing said plurality of second transposons inserted into said plurality of amplified first end truncated DNA inserts with Phage P1, and wherein Cre protein is expressed by Phage P1 during said infection;
• n. Truncating an opposite end portion of each of said plurality of amplified first end truncated DNA inserts, wherein each of said amplified first end truncated DNA inserts contains one of said inserted second transposons and creating a plurality of doubly end truncated DNA inserts with a new opposite end;
• o. Packaging said plurality of doubly end truncated DNA inserts in said vector into said P1 Phage head;
• p. Using said P1 Phage head to infect a second colony of fresh NS3516 cells to amplify said plurality of doubly end truncated DNA inserts by selecting on LB agar plates containing said first antibiotic and a second antibiotic whose resistance gene exists in said second transposon, and thereby amplifying a plurality of doubly end truncated DNA inserts in a plurality of NS3516 cell colonies generated from said second colony of fresh NS3516 cells;
• q. Isolating said amplified plurality of different doubly end truncated DNA inserts in said vector to obtain a plurality of colonies, each colony with the same opposite end truncation from said second colony of fresh NS3516 cells;
• and
• r;
• Using a second synthetic DNA primer to sequence said amplified plurality of new opposite end portions of said different doubly end truncated DNA inserts.

A method of selecting transpositions using phage P1 headful packaging instead of using an antibiotic resistance gene within a transposon comprising:

• a. Constructing a plurality of plasmids of identical sequence each containing a first transposon carrying either a wild type loxP or a mutant loxP site;
• b. Transforming said plurality of first transposon containing plasmids into cells, each containing a DNA insert in said vector carrying a first antibiotic resistance gene;
• c. Inducing each of said plurality of first transposons to insert into said DNA inserts in said cells;
• d. Infecting said cells containing said plurality of first transposons inserted into said DNA inserts with Phage P1, and wherein Cre protein is expressed by Phage P1 during said infection;
• e. Truncating first end portions of each of said DNA inserts, wherein each of said DNA inserts contains one of said inserted first transposons, and thereby creating a plurality of first end truncated DNA inserts with new first ends;
• f. Packaging said first end truncated DNA inserts into a P1 Phage head;
• g. Using said P1 Phage head to infect a first colony of fresh NS3516 cells to amplify said first end truncated DNA inserts by selecting on LB agar plates containing a first antibiotic whose resistance gene occurs in said vector, and thereby amplifying said plurality of first end truncated DNA inserts in a plurality of NS3516 cell colonies generated from said first colony of fresh NS3516 cells;
• h. Isolating said amplified plurality of different first end truncated DNA inserts in said vector to obtain a plurality of colonies, each colony with the same end truncation, from said first colony of fresh NS3516 cells;
• and
• i. Using a first synthetic DNA primer to sequence said amplified plurality of new first end portions of said amplified plurality of different first end truncated DNA inserts.