09/09/13

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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)280/260260/280
TOD X131.686.51.871.92
TOD X231.758.71.891.26
TOD X332.756.61.911.88
TOD X431.655.81.881.65
TOD X531.744.41.871.78
TOD X631.770.51.881.88
TOD F132.439.61.911.59
TOD F233.749.41.911.65
TOD F332.347.41.891.71
TOD F431.643.71.941.73
TOD F536.5571.891.50
TOD F63859.41.891.61

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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.