Team:UNICAMP-EMSE Brazil/Notebook/2 September 2011

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May
MTWTFSS
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30 31
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MTWTFSS
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MTWTFSS
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August
MTWTFSS
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29 30 31
September
MTWTFSS
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26 27 28 29 30
October
MTWTFSS
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31


Electrophoresis Gel 1, 02/09/2011.
Gel 1: SoxS+RBS+IL10+HlyA+T C2 (nd)/ SoxS+RBS+IL10+HlyA+T C1/ SoxS+RBS+IL10+HlyA+T C2/ SoxS+RBS+IL10+HlyA+T C3/ SoxS+RBS+IL10+HlyA+T C4/ SoxS+RBS+GFP+HlyA+T C2 (nd)/ SoxS+RBS+GFP+HlyA+T C1/ SoxS+RBS+GFP+HlyA+T C2/ SoxS+RBS+GFP+HlyA+T C3/ SoxS+RBS+GFP+HlyA+T C4/ SoxS+RBS+HlyB+HlyD+TolC+T (nd)/ SoxS+RBS+HlyB+HlyD+TolC+T

02 September 2011

Checking digestion (E-P) of mini-prep constructs

Objective:

  • Checking digestion (E-P) of mini-prep constructs.

Samples:

Electrophoresis Gel 2, 02/09/2011.
Gel 2: flHDC+RBS+HlyB+hlyD+TolC+T C4(nd)/ flHDC+RBS+HlyB+hlyD+TolC+T C2/ flHDC+RBS+HlyB+hlyD+TolC+T C3/ flHDC+RBS+HlyB+hlyD+TolC+T C4/ RBS+GFP+HlyA+T C2 (nd)/ RBS+GFP+HlyA+T C1/ RBS+GFP+HlyA+T C2/ flHDC+RBS+IL12 C5 (nd)/ flHDC+RBS+IL12 C1/ flHDC+RBS+IL12 C2/ flHDC+RBS+IL12 C5.
  • Gel 1:
  • SoxS+RBS+IL10+HlyA+T C2 (nd)/ SoxS+RBS+IL10+HlyA+T C1/ SoxS+RBS+IL10+HlyA+T C2/ SoxS+RBS+IL10+HlyA+T C3/ SoxS+RBS+IL10+HlyA+T C4/ SoxS+RBS+GFP+HlyA+T C2 (nd)/ SoxS+RBS+GFP+HlyA+T C1/ SoxS+RBS+GFP+HlyA+T C2/ SoxS+RBS+GFP+HlyA+T C3/ SoxS+RBS+GFP+HlyA+T C4/ SoxS+RBS+HlyB+HlyD+TolC+T (nd)/ SoxS+RBS+HlyB+HlyD+TolC+T
  • Gel 2:
  • flHDC+RBS+HlyB+hlyD+TolC+T C4(nd)/ flHDC+RBS+HlyB+hlyD+TolC+T C2/ flHDC+RBS+HlyB+hlyD+TolC+T C3/ flHDC+RBS+HlyB+hlyD+TolC+T C4/ RBS+GFP+HlyA+T C2 (nd)/ RBS+GFP+HlyA+T C1/ RBS+GFP+HlyA+T C2/ flHDC+RBS+IL12 C5 (nd)/ flHDC+RBS+IL12 C1/ flHDC+RBS+IL12 C2/ flHDC+RBS+IL12 C5.


  • Ladder:
  • 100-10.000 bp (Fermentas)
  • Gel Agarose concentration:
  • 1,0%


  • Results:

Gene Total size (gene+vector) Linear vector size Gene size Result
SoxS+RBS+IL10+HlyA+T C1 4270 bp 3205 bp 1065 bp Did not digest
SoxS+RBS+IL10+HlyA+T C2 4270 bp 3205 bp 1065 bp Did not digest
SoxS+RBS+IL10+HlyA+T C3 4270 bp 3205 bp 1065 bp Did not digest
SoxS+RBS+IL10+HlyA+T C4 4270 bp 3205 bp 1065 bp Did not digest
SoxS+RBS+GFP+HlyA+T C1 4365 bp 3205 bp 1160 bp OK
SoxS+RBS+GFP+HlyA+T C2 4365 bp 3205 bp 1160 bp Smaller bands
SoxS+RBS+GFP+HlyA+T C3 4365 bp 3205 bp 1160 bp Smaller bands
SoxS+RBS+GFP+HlyA+T C4 4365 bp 3205 bp 1160 bp OK
SoxS+RBS+HlyB+HlyD+TolC + T 8257 bp 3205 bp 5052 bp OK
fLHDC+RBS+HlyB+HlyD+TolC + T C2 8280 bp 3205 bp 5075 bp OK
fLHDC+RBS+HlyB+HlyD+TolC + T C3 8280 bp 3205 bp 5075 bp OK
fLHDC+RBS+HlyB+HlyD+TolC + T C4 8280 bp 3205 bp 5075 bp OK
RBS+GFP+HlyA+T C1 4262 bp 3205 bp 1057 bp Did not digest
RBS+GFP+HlyA+T C2 4262 bp 3205 bp 1057 bp Did not digest
fLHDC+RBS+IL12 C1 4584 bp 2800 bp 1784 bp OK
fLHDC+RBS+IL12 C1 4584 bp 2800 bp 1784 bp Diluted (?)
fLHDC+RBS+IL12 C1 4584 bp 2800 bp 1784 bp No bands



  • Checking digestion again:
  • SoxS+RBS+IL10+HlyA+T and RBS+GFP+HlyA+T
  • Real digestion for ligation:
  • fLHDC+RBS+IL12 C1 with HlyA+T
  • Ready Biobricks:
  • fLHDC+RBS+HlyB+HlyD+TolC + T / SoxS+RBS+GFP+HlyA+T / SoxS+RBS+HlyB+HlyD+TolC + T.

  • Ready to test:
  • Device that produces and secrete IL10 !  :)




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