Team:UNICAMP-EMSE Brazil/Notebook/25 August 2011

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Notebook

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May
MTWTFSS
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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
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
July
MTWTFSS
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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, 25/08/2011.
Gel 1:RBS+TolC+T C1 (d)/ RBS+TolC+T C1 (nd)/ RBS+TolC+T C2 (d)/ RBS+TolC+T C2 (nd)/ RBS+TolC+T C3 (d)/ RBS+TolC+T C3 (nd)/ RBS+TolC+T C4 (d)/ RBS+TolC+T C4 (nd).

25 August 2011

RBS+TolC+T and RBS+SoxR_T

Objective:

  • Checking “RBS+TolC+T” and “RBS+SoxR+T” purified by Mini-prep and digested with E-P.
Electrophoresis Gel 2, 25/08/2011.
RBS+SoxR+T C1 (d)/ RBS+SoxR+T C1 (nd)/ RBS+SoxR+T C2 (d)/ RBS+SoxR+T C2 (nd)/ RBS+SoxR+T C3 (d)/ RBS+SoxR+T C3 (nd)/ RBS+SoxR+T C4 (d)/ RBS+SoxR+T C4 (nd).
  • Ladder:
  • Bio-Rad 100bp – 10.000bp
  • Samples:
  • Gel 1: RBS+TolC+T C1 (d)/ RBS+TolC+T C1 (nd)/ RBS+TolC+T C2 (d)/ RBS+TolC+T C2 (nd)/ RBS+TolC+T C3 (d)/ RBS+TolC+T C3 (nd)/ RBS+TolC+T C4 (d)/ RBS+TolC+T C4 (nd)
  • Gel 2: RBS+SoxR+T C1 (d)/ RBS+SoxR+T C1 (nd)/ RBS+SoxR+T C2 (d)/ RBS+SoxR+T C2 (nd)/ RBS+SoxR+T C3 (d)/ RBS+SoxR+T C3 (nd)/ RBS+SoxR+T C4 (d)/ RBS+SoxR+T C4 (nd)

OBS:

  • D=digested
  • Nd= non-digested


  • Results:

Gene Total size (gene+vector) Linear vector size Gene size Result
RBS+TolC+T ~4880 pb 3300 pb 1675 pb OK: complete digestion
RBS+SoxR+T ~3823 pb 3300 pb 618 pb Only C4 did not digested.
C1 and C3 has a gene fragment near 500 pb
and C2 has one near 700 bp.
Wich one is the correct?



Isolate digested biobricks from their vectors

Electrophoresis Gel 3, 25/08/2011.
  • Ladder:
  • Bio-Rad 100bp – 10.000bp

Objective:

  • isolate digested biobricks from their vector (volume of ladder applied 6uL; digestion sample:40uL digested sample+4uL buffer)
  • Samples:
  • 1: RBS+TolC+terminator (digested) ;
  • 2: RBS+TolC+terminator (non-digested);
  • 3: RBS+IL10 (digested);
  • 4: RBS+IL10 (non-digested);
  • 5: RBS+IL12 (digested);

6 :RBS+IL12 (Non-digested);

  • 7: RBS+SoxR+Terminator (digested);
  • 8: RBS+SoxR+Terminator (non-digested).
  • 9: RBS+SoxR+Terminator (digested);


  • Results:

Gene Total size (gene+vector) Linear vector size Gene size Result
RBS+TolC+terminator 4880 pb 3300 pb 1675 pb Bright higher band
RBS+IL10 3345 pb 2800 pb 545 pb Didn’t work
RBS+IL12 4458 pb 2800 pb 1658 pb OK
RBS+SoxR+Terminator ~3823 pb 3300 pb 1618 pb OK
RBS+SoxR+Terminator ~3823 pb 3300 pb 1618 pb OK



Electrophoresis Gel 4, 25/08/2011.
  • Ladder:
  • Bio-Rad 100bp – 10.000bp

Objective:

  • quantification of the purified bands from the previous electrophoresis
  • Samples:
  • 1: RBS+TolC+terminator ;
  • 2: RBS+IL12 ;
  • 3: RBS+SoxR+Terminator
  • Results:
  • Their concentration seems quite good!




Digestions recipes

  • RBS+TolC+Terminator (E-X)
  • 29ul - RBS+TolC+Terminator
  • 8ul - 10X Tango Buffer
  • 1ul - EcoRI
  • 2ul - XbaI
  • TOTAL = 40ul
  • RBS+IL-10 (X-P)
  • 32ul - RBS+IL-10
  • 4ul - 10X Tango Buffer
  • 1.4ul - XbaI
  • 2.6ul - PstI
  • TOTAL = 40ul
  • RBS+IL-12 (X-P)
  • 32ul - RBS+IL-12
  • 4ul - 10X Tango Buffer
  • 1.4ul - XbaI
  • 2.6ul - PstI
  • TOTAL = 40ul
  • RBS+SoxR (X-P)
  • 32ul - RBS+SoxR
  • 4ul - 10X Tango Buffer
  • 1.4ul - XbaI
  • 2.6ul - PstI
  • TOTAL = 40ul


Next Tasks:

  1. Incubate reactions at 37°C for 3-4hours.
  2. Electrophoresis (loading the whole digestions volume - join two or more wells in the gel)
  3. Purify the bands
  4. Electrophoresis to confirm (load 5ul of gel purified digestion)