From 2013.igem.org

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Dissolution of polystyrene

  • a 20ml solution of 50% Limonene was made to determine if the dissolution of polystyrene from 09/09/13 would occur faster.
  • as predicted, the 8cm x 5cm x 2cm PS block was completely dissolved in 9 minutes.

Gel electrophoresis

  • The samples from the double digest on 09/09/13 were run to confirm if the digestion worked.
  • From the results, we would also be able to distinguish between cells that have been successfully transformed with the ligated pSB1C3/TOD genes from those with the recircularised pSB1C3.

Results of the Gel electrophoresis


Doubledigest TODX 10013.jpg

Wells order: TODX1 (SpeI/XbaI), TODX1 (EcoRI/PstI); TODX2 (SpeI/XbaI), TODX2 (EcoRI/PstI); TODX3 (SpeI/XbaI), TODX3 (EcoRI/PstI); TODX4 (SpeI/XbaI), TODX4 (EcoRI/PstI); TODX5 (SpeI/XbaI), TODX5 (EcoRI/PstI); TODX6 (SpeI/XbaI), TODX6 (EcoRI/PstI).

The expected results were that the we would see the TODX genes drop off in separate band in all the digestions in its respective size (600bp). However as it can be seen in the picture above only the samples cut with SpeI and XbaI had two fragments and they are the wrong size (~ 1Kb). The samples TODX (1, 2 and 6) were sent to be sequenced before the digestions and it showed that the pSB1C3 plasmid re-circularized. This is because the two enzymes have complementary sticky ends.


Double digestions TODF 100913.jpg

Wells order: TODF1 (SpeI/XbaI), TODF1 (EcoRI/PstI); TODF2 (SpeI/XbaI), TODF2 (EcoRI/PstI); TODF3 (SpeI/XbaI), TODF3 (EcoRI/PstI); TODF4 (SpeI/XbaI), TODF4 (EcoRI/PstI); TODF5 (SpeI/XbaI), TODF5 (EcoRI/PstI); TODF6 (SpeI/XbaI), TODF6 (EcoRI/PstI).

This shows the same results as TODX above.


Double digestion TOBG 100913.jpg

Wells order: TOBG1 (SpeI/XbaI), TOBG1 (EcoRI/PstI); TOBG2 (SpeI/XbaI), TOBG2 (EcoRI/PstI); TOBG3 (SpeI/XbaI), TOBG3 (EcoRI/PstI); TOBG4 (SpeI/XbaI), TOBG4 (EcoRI/PstI); TOBG5 (SpeI/XbaI), TOBG5 (EcoRI/PstI); TOBG6 (SpeI/XbaI), TOBG6 (EcoRI/PstI).

This shows the same results as TODX above.

Nanodrop of the pGEM-T plasmids that were ligated with the TOD operon genes and the pSB1C3 backbone

SampleVolumeConcentration ng/ul260/280260/230
TodX 125.8 37.4 1.84 1.52
TodX 235 61.3 1.82 1.74
TodF 137.8 99.4 1.831.99
TodF 222.725.71.761.60
ToBG 131.4 51.7 1.841.57
pSB1C3 110.331.81.651.44
pSB1C3 11940.91.811.71

Double Digestions of the plasmids above

SampleDNA volumeBufferXbaIPstIdH2O to reach final volume of 60 ul