IGEM:Indian Institue of Technology Madras/2009/Notebook/PLASMID - Plasmid Locking Assembly for Sustaining Multiple Insert DNA/2009/09/12

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Entry title

  • Primers received.
  • PCRs done:
  • Template--Forward primer-----------Reverse primer
  • 1 S03879----BB Pre +J23119 overhang--BB suff + overhang
  • 2 S03879----BB Pre + Overhang--------BB Suff + overhang
  • 3 I13502----BB Pre + Overhang--------BB Suff + overhang
  • 4 I13507----BB Pre + Overhang--------BB Suff + overhang
  • 5 K145151---BB Pre + Overhang--------BB Suff + overhang
  • 6 K145151---BB Pre + Overhang--------BB Suff + overhang
  • gel 1 order: 1 through 6(Unpurified); 1 through 6 (purified)
  • PCR protocol used:
  • 95°C for 2 mins
  • 95°C for 30 sec
  • (Tm-5)°C for 30 sec
  • 72°C for Te for 1 min
  • Repeat steps 2 to 4 (30 times)
  • 72°C for 30 min.
  • 4°C for ever
  • PCR purification done.
  • Restrictions:
  • no:---Product---Enzyme
  • 1 -- PCR1 -- S
  • 2 -- PCR2 -- S
  • 3 -- PCR3 -- X
  • 4 -- PCR4 -- X


  • Ligations:
  • Lig---RD#S
  • 1---1+3
  • 2 --1+4
  • 3---2+3
  • 4---2+4
  • Ligations 1;2;3;4 were PCRed with BB pre and Suff primers. They were PCR purified. Restricted with E and P. Ligated them with the vectors which were digested with E and P.
  • gel 2 Order: I13507(1); I13507(2); I13502(1); I13502(2); S03879(dye from kit); S03879(1); S03879(2); LADDER.
  • Ligations:
  • J23119(S/P)(1) + E0430(X/P)(1)----SA(Chl)
  • J23119(S/P)(1) + I13504(X/P(1)----SA(Chl)
  • K145151(E/S) + E0420(E/X)----SA(Chl)
  • J13002(S/P)(1) + E0020----SA(Chl)
  • J13002(S/P)(1) + E0420----SA(Chl)
  • J13002(S/P)(2) + E0020----SA(Chl)
  • J13002(S/P)(2) + E0420----SA(Chl)
  • S03879(S/P)(1) + I13507----SA(Chl)
  • E0430(E/P) + 1C3
  • I13504(E/P) + 1C3
  • I13522(E/P) + 1C3
  • Gel run for all digests:
  • Gel 3 order:
  • ROW1: E0420(E/X); I13504(X/P)(2); S03879(S/P)(1); J13002(S/P)(1); E0020(X/P); I13504(X/P)(1); I13504(E/P)(2); PSB1C3(E/P); I13504(E/P)(1); J13002(S/P)(2); LADDER; E0430(E/P)(1); E0430(E/P)(2); J23119(S/P); J13522(E/P); I13507(X/P).
  • ROW2: I13522(1A2)(1); I13522(E/P)(1); I13522(2)(1A2); I13522(E/P)(2); I13504(1A2)(1); I13504(E/P)(1); LADDER; I13504(1A2)(1); I13504(E/P)(2); E0430(1A2)(1); E0430(E/P)(1); E0430(1A2)(2); E0430(E/P)(2).