Team:UNICAMP-EMSE Brazil/Notebook/7 September 2011

07 September 2011

Objective:

• Electrophoresis to cut and purify bands

Samples:

• Gel 1:

• flhDC_RBS_IL12(C1)(E - S) / RBS_GFP_HlyA_Term(C2)(X - P)

• Ladder:

• 100-10.000 bp (Fermentas)

• Gel Agarose concentration:

• 1,0%

• Expected sizes:

• 1784bp / ~1050bp - both are OK

Objective:

• Electrophoresis to cut and purify bands

Samples:

• Gel 1:

• flhDC_RBS_IL12(C1) / RBS_GFP_HlyA_Term(C2)

• Ladder:

• 100-10.000 bp (Fermentas)

• Gel Agarose concentration:

• 1,0%

Digestions recipes for gel purification:

• RBS+GFP+HlyA+T (X-P)

• 32ul - RBS+GFP+HlyA+T miniprep (C2)
• 4ul - 10X Tango Buffer
• 1.4ul - XbaI
• 2.6ul - PstI
• TOTAL = 40ul

• flhDC+RBS+IL-12 (E-S)

• 31ul - flhDC+RBS+IL-12 miniprep (did on 06/09/2011)
• 4ul - 10X Buffer R
• 1ul - EcoRI
• 4ul - SpeI
• TOTAL = 40ul

• flhDC (S-P)

• 32ul - flhDC miniprep
• 4ul - 10X Tango Buffer
• 1.4ul - SpeI
• 2.6ul - PstI
• TOTAL = 40ul

• HlyA+T (E-X)

• 29ul - HlyA+T miniprep
• 8ul - 10X Tango Buffer
• 1ul - EcoRI
• 2ul - XbaI
• TOTAL = 40ul

Tasks:
Incubate reactions at 37°C for 4hours.
Electrophoresis (loading the whole digestions volume - join two wells in the gel)
Purify the bands

Electrophoresis to confirm (load 5ul of gel purified digestion)