Team:UNICAMP-EMSE Brazil/Notebook/30 August 2011

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May
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31
June
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    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
July
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
August
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31
September
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30
October
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31


Electrophoresis Gel 1, 30/08/2011.
Gel 1: RBS+HlyD+TolC+Terminator (E-X) C1/ RBS+HlyD+TolC+Terminator (E-X) C2/ RBS+HlyD+TolC+Terminator (E-X) C1a/ RBS+HlyD+TolC+Terminator (E-X) C2a/ HlyA+Terminator (E-X) C4/ HlyA+Terminator C3 (E-X)/ HlyA+Terminator C2 (E-X)/SoxS+RBS+HlyB (E-S) C1/ SoxS+RBS+GFP (E-S) C2/flHDC+RBS+HlyB (E-S) C2/ SoxS+RBS+IL 10 (E-S) C2

30 August 2011

Finishing the first two biobricks!!!

Objective:

  • Digestion of constructs to purify and ligate them.

Samples:

  • Gel 1:
  • RBS+HlyD+TolC+Terminator (E-X) C1/ RBS+HlyD+TolC+Terminator (E-X) C2/ RBS+HlyD+TolC+Terminator (E-X) C1a/ RBS+HlyD+TolC+Terminator (E-X) C2a/ HlyA+Terminator (E-X) C4/ HlyA+Terminator C3 (E-X)/ HlyA+Terminator C2 (E-X)/SoxS+RBS+HlyB (E-S) C1/ SoxS+RBS+GFP (E-S) C2/flHDC+RBS+HlyB (E-S) C2/ SoxS+RBS+IL 10 (E-S) C2
  • Ladder:
  • 100-10.000 bp (Fermentas)
  • Gel Agarose concentration:
  • 1,0%


  • Results:

Construction Total size (gene+vector) (bp) Linear vector size (bp) Gene size (bp) Result
RBS+HlyD+TolC+Terminator C1 6376 3205 3171 Run more, strange digested band. We supposed to have just linearized vector.
RBS+HlyD+TolC+Terminator C2 6376 3205 3171 Run more, strange digested band. We supposed to have just linearized vector.
RBS+HlyD+TolC+Terminator C1a 6376 3205 3171 Run more, strange digested band. We supposed to have just linearized vector.
RBS+HlyD+TolC+Terminator C2a 6376 3205 3171 Run more, strange digested band. We supposed to have just linearized vector.
HlyA+Terminator C4 3527 3205 322 Run more, strange digested band. We supposed to have just linearized vector
HlyA+Terminator C3 3527 3205 322 Run more, seems OK.
HlyA+Terminator C2 3527 3205 322 Run more, strange digested band. We supposed to have just linearized vector
SoxS+RBS+HlyB C1 5086 2800 2286 Ok, run more
SoxS+RBS+GFP C2 3535 2800 838 Ok
flHDC+RBS+HlyB C1 5109 2800 2309 Ok, run more
flHDC+RBS+GFP C1 3661 2800 861 Ok
SoxS+RBS+IL 10 C2 3543 2800 743 Ok




We cut SoxS+RBS+IL10 (E-S) and SoxS+RBS+ GFP (E-S), but during the cutting we lost the last one :(
The other samples we left running for more time. Problem with flHDC+RBS+HlyB C1 ! Lost it too! We could save just SoxS+RBS+HlyB and SoxS+RBS+IL10 to purification!!!!


Electrophoresis Gel 2, 30/08/2011.
Gel 2: RBS+HlyD+TolC+Terminator (E-X) C1/ RBS+HlyD+TolC+Terminator (E-X) C2/ RBS+HlyD+TolC+Terminator (E-X) C1a/ RBS+HlyD+TolC+Terminator (E-X) C2a/ HlyA+Terminator (E-X) C4/ HlyA+Terminator C3 (E-X)/ HlyA+Terminator C2 (E-X)/SoxS+RBS+HlyB (E-S) C1/ SoxS+RBS+GFP (E-S) C2/flHDC+RBS+HlyB (E-S) C2/ SoxS+RBS+IL 10 (E-S) C2.

Same electrophoresys but after a longer period run:

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

Samples:

  • Gel 2:
  • HlyA+Terminator C1/ HlyA+Terminator C2/ HlyA+Terminator C3/ HlyA+Terminator C4/ Const. Promoter+ RBS+SoxR+ Terminator C1/ Const. Promoter+ RBS+SoxR+ Terminator C2/ SoxS+RBS+IL 10 C1/ SoxS+RBS+IL 10 C2/ flHDC+IL 12 C1/ flHDC+IL 12 C2/ RBS+HlyD+TolC+Terminator C2a/ RBS+HlyD+TolC+Terminator C1a

Results:

  • By the pattern of the bands observed in the gel, somebody made a mistake and used E-S to digest RBS+HlyD+TolC+T and HlyA+T instead of E-X to linearize.

Bad labday!!! Murphy is here !!! Murphy's law confirmed!!!


Checking digestion (E-P) of flHDC+RBS+IL12 and flHDC+RBS+GFP mini-preped today:

Objective:

Electrophoresis Gel 3, 30/08/2011.
Gel 3: SoxS+RBS+HlyB (E-S)/ SoxS+RBS+IL10 (E-S)/ flHDC+RBS+GFP C5/ flHDC+RBS+GFP C6/ flHDC+RBS+GFP C7/ flHDC+RBS+IL 12 C5/ flHDC+IL 12 C6
  • Checking digestion (E-P) of flHDC+RBS+IL12 and flHDC+RBS+GFP mini-preped today and quantification of SoxS+RBS+HlyB (E-S) and SoxS+RBS+IL10 (E-S) purified from the latter gel
  • Ladder:
  • 100-10.000 bp (Fermentas)
  • Gel Agarose concentration:
  • 1,0%
  • Gel 3:
  • SoxS+RBS+HlyB (E-S)/ SoxS+RBS+IL10 (E-S)/ flHDC+RBS+GFP C5/ flHDC+RBS+GFP C6/ flHDC+RBS+GFP C7/ flHDC+RBS+IL 12 C5/ flHDC+IL 12 C6



  • Results:

Gene Total size (gene+vector) Linear vector size Gene size Result
flHDC+RBS+GFP C5 3661 bp 2800 bp 861 bp No visible bands
flHDC+RBS+GFP C6 3661 bp 2800 bp 861 bp OK
flHDC+RBS+GFP C7 3661 bp 2800 bp 861 bp No visible band of GFP
flHDC+RBS+IL 12 C5 4584 bp 2800 bp 1784 bp Where is the vector?
flHDC+IL 12 C6 4584 bp 2800 bp 1784 bp Where is the vector?



OBS:--Inoculate more SoxS+RBS+GFP (c2), HlyA+T (C3) and RBS+HlyD+RBS+TolC+Terminator (C1a), flHDC+RBS+HlyB (C1) , SoxS+RBS+IL10 C2, to digest more tomorrow.






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