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Team:UNICAMP-EMSE Brazil/Notebook/25 August 2011
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Notebook
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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.
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
- 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 |
- 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:
- Incubate reactions at 37°C for 3-4hours.
- Electrophoresis (loading the whole digestions volume - join two or more wells in the gel)
- Purify the bands
- Electrophoresis to confirm (load 5ul of gel purified digestion)
