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 S03879BB Pre +J23119 overhang--BB suff + overhang
 * 2 S03879BB Pre + OverhangBB Suff + overhang
 * 3 I13502BB Pre + OverhangBB Suff + overhang
 * 4 I13507BB Pre + OverhangBB Suff + overhang
 * 5 K145151---BB Pre + OverhangBB Suff + overhang
 * 6 K145151---BB Pre + OverhangBB 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) + E0020SA(Chl)
 * J13002(S/P)(1) + E0420SA(Chl)
 * J13002(S/P)(2) + E0020SA(Chl)
 * J13002(S/P)(2) + E0420SA(Chl)
 * S03879(S/P)(1) + I13507SA(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).


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