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From 2013.igem.org

Miniprep of overnight broth

• Following on from 06/09/13 result,overnight broth was prepared yesterday from the single colonies that had grown on each of the 2 plates shown on 06/09/13.
• The broth, which contains cells that have been transformed with pSB1C3/TOD genes, was minipreped using the Omega Bio-Tek kit.
• This would enable us to confirm which cells have the recircularised plasmid and those with the ligated product.

Measurement of DNA concentration

Samples	Volume (ul)	Conc (ng/ul)	280/260	260/280
TOD X1	31.6	86.5	1.87	1.92
TOD X2	31.7	58.7	1.89	1.26
TOD X3	32.7	56.6	1.91	1.88
TOD X4	31.6	55.8	1.88	1.65
TOD X5	31.7	44.4	1.87	1.78
TOD X6	31.7	70.5	1.88	1.88
TOD F1	32.4	39.6	1.91	1.59
TOD F2	33.7	49.4	1.91	1.65
TOD F3	32.3	47.4	1.89	1.71
TOD F4	31.6	43.7	1.94	1.73
TOD F5	36.5	57	1.89	1.50
TOD F6	38	59.4	1.89	1.61

Double Digestion of pSB1C3/TOD genes

• The pSB1C3/TOD genes were double digested with (XbaI and SpeI) & (EcoRI and PstI)
• The master mix for the (XbaI and SpeI) was;

60ul of cut smart buffer 5ul of XbaI 10ul of SpeI 273ul of 5M Tris

• The master mix for the (EcoRI and PstI) was;

60ul of cut smart buffer 5ul of XbaI 5ul of SpeI 278ul of 5M Tris

All 36 tubes were incubated overnight in 37 degrees room.

• Tomorrow the plasmids that were isolated from the miniprep would be run on a 1% agarose gel to distinguish between cells that have the pSB1C3/TOD genes from those that have the recircularised pSB1C3 vector.