Cloning of T7 promoter + rbs + GFP + terminator 2009/6/24 10:50 Miniprep the plasmids containing T7 promoter, standard parts 1-12M and 1-12O. 1-12M: medium rbs + GFP coding gene + terminator 1-12O: strong rbs + GFP coding gene + terminator 12:30 Digest the plasmids of T7 promoter, 1-12M and 1-12O Digestion System for plasmids of T7 promoter: 50μL Plasmids: 30μL; Spe1: 1μL; Buffer 1*H: 5μL. Digestion System for plasmids of 1-12M and 1-12O: 50μL Plasmids: 30μL; Xba1: 1μL; Buffer 1*M: 5μL. 12:50 Start to digest 14:20 Add CIAP into the system of vector digestion. Buffer: 5μL; Enzyme: 1μL. 15:20 Electrophoresis to recycle the vectors and inserts of digestion. The order of the samples: marker, plasimid control of T7 promoter, T7 promoter, 1-12M, 1-12O. Recycle the fragments (vector) of T7 promoter and the inserts of 1-12M, 1-12O. The fragment of T7 promoter is weird, for the linear plasmids ran even faster than the round ones. Decide to do some controls and tests 2009/6/25 10:00 Miniprep the plasmids containing T7 promoter, standard parts 1-12M and 1-12O. 11:00 Electrophoresis of these plasmids
The order and the amount of the samples: marker 10μL, 1-12M 10μL, T7 promoter 10μL, 1-12O 10μL. The circular plasmids present at smaller places of their real size, which demonstrates that the plasmids are normal. 15:30 Single digestion of the plasmids of the T7 promoter to test the efficiency of the single digestion. Digestion System 1: 20μL; Plasmids: 1μL; Buffer 1*H: 2μL; dd H2O: 16μL. Digestion System 2: 20μL; Plasmids: 5μL; Buffer 1*H: 2μL; dd H2O: 12μL. 16:00 Strart to digest. 20:00 Electrophoresis to test the single digestion. The order and the amount of the samples: marker 10μL,plasmids of T7 promoter 10μL, digesion product of system 1 10μL, digesion product of system 2 10μL.
The single digestion product is slower than the cicular ones, suggesting that the single digestion is correct and the efficiency is OK. 21:30 Recycle the fragments. The products of recycling are thrown away by chance by ZHQ. 2009/6/28 14:00 Miniprep the plasmids of T7 promoter, 1-12M, 1-12O. The concentration of the plasmids: T7 promoter: 305ng/μL; 1-12M: 280ng/μL; 1-12O: 295ng/μL. 15:00 Digest the plasmids of T7 promoter, 1-12M and 1-12O Digestion System for plasmids of T7 promoter: 20μL Plasmids: 3μL; Buffer 1*H: 2μL; dd H2O: 14μL. Digestion System for plasmids of 1-12M and 1-12O: 20μL Plasmids: 4μL; Xba1: 1μL; Buffer 1*M: 2μL; dd H2O: 12μL. 17:10 Start to digest. 22:00 Double digestion of plasmid of T7 promoter. Digestion System for plasmids of T7 promoter: 20μL Plasmids: 3μL; Xba1: 1.5μL; Pst1: 1.5μL;
Buffer 1*M: 2μL; dd H2O: 12μL. 23:25 Electrophoresis to test the digestion efficiency (17:10). The order and the amount of the samples: marker 10μL, plasmids of T7 promoter 10μL, digestion products of T7 promoter 10μL, plasmids of 1-12M 10μL; digestion products of 1-12M, plasmids of 1-12O, digestion products of 1-12O. The plasmids have been cut downright and are suitable for recycling. 2009/6/29 1:30 Recycle the fragments of single digestion of T7 promoter. Electrophoresis to recycle the fragments of 1-12M and 1-12O The order and the amount of the samples: marker 10μL, plasmids of 1-12M 10μL; digestion products of 1-12M, plasmids of 1-12O, digestion products of 1-12O.
2:00 Single digestion of the T7 promoter fragments Fragments: 10μL; Spe1: 1.5μL; Buffer: 2μL; ddh2o: 5.5μL; 2:20 Start to digest. 14:00 Electrophoresis to test the efficiency of double digestion The order and the amount of the samples: marker 10μL, plasmids of T7 promoter 10μL, digestion products of T7 promoter 10μL. 15:00 Recycle the fragments of T7 promoter, both double digestion and single digestion. 15:30 Add CIAP into another system of T7 promoter. 16:00 Electrophoresis to recycle the fragments of 1-12M and 1-12O The order and the amount of the samples: marker 5μL, plasmids of T7 promoter 10μL, digestion products of T7 promoter 10μL. 16:50 Recycle the double digestion products of T7 promoter. 20:30 Ligation: T7 promoter + GFP T7 promoter Double digestion with CIAP Double digestion without CIAP Single digestions separated GFP 1-12M Medium rbs + GFP coding gene + terminator 1-12O Strong rbs + GFP coding gene + terminator System of Ligation 7:1 (μl) 3:1 (μl) Control (μl)
Vector 1 1 1 Insert 7 3 0 T4 ligase 1 1 1 Buffer 1 1 1 ddh2o 0 4 7 21:00 Start to ligase 2009/6/30 10:30 Transformation: transform the ligation to DH5α 2009/7/1 4:00 Proportion 3:1 is enough for the ligation and CIP is necessary for the double digestion. Choose the plates of double digestion with CIAP to pick the single clones and each plate pick 5 clones. Incubate them at 37. 17:30 Mimiprep the plasmids of ligation 20:00 Electrophoresis to test the ligation products The order and the amount of the samples: marker 5μL, plasmids of T7 promoter 2μL, ligation products (1-12M) 2μL, ligation products (1-12O) 2μL. O3 is weird, others are correct. 21:00 Digestion to test the ligation products Digestion System: 20μL; Plasmids: 1μL; EcoR1: 1μL;
Buffer: 2μL; ddh2o: 15μL; 21:50 Start to digest. 2009/7/2 1:30 Electrophoresis to test the ligation products The order and the amount of the samples: marker 5μL, plasmids of T7 promoter 2μL, ligation products (1-12M) 2μL, ligation products (1-12O) 2μL. The results demonstrate that all the ligation products are correct except O3, which the inserts has been linked 3 times into the backbone.