Team:UNICAMP-EMSE Brazil/Notebook/19 September 2011

Ligations:

=> IL-10 (~550bp) and pSB1C3 (~2000bp) (ratio for ligation 3:1)

3.8 ul - H20
20 ul - digested IL-10 (~150 ng )
2.2 ul - digested vector [pSB] (~50 ng)
3 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________ 30 ul => TOTAL

=> IL-12 (~1660bp) and pSB1C3 (~2000bp) (ratio for ligation 3:1)

3.9 ul - H20
15 ul - digested IL-12 (~180 ng )
2.6 ul - digested vector [pSB] (~60 ng)
2.5 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________ 25 ul => TOTAL

=> HlyD (~1500bp) and pSB1C3 (~2000bp) (ratio for ligation 3:1)

3.9 ul - H20
15 ul - digested HlyD (~180 ng )
2.6 ul - digested vector (~60 ng)
2.5 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________ 25 ul => TOTAL

=> QseB/C (~2100bp) and pSB1C3 (~2000bp) (ratio for ligation 1:1)

10.1 ul - H20
4.3 ul - digested QseB-QseC (~60 ng )
2.6 ul - digested vector [pSB] (~60 ng)
2 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________ 20 ul => TOTAL

=> HlyB (~2200bp) and pSB1C3 (~2000bp) (ratio for ligation 1:1)

10.9 ul - H20
3.5 ul - digested HlyB (~60 ng )
2.6 ul - digested vector [pSB] (~60 ng)
2 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________ 20 ul => TOTAL

=> TolC (1580bp) and pSB1C3 (~2000bp) (ratio for ligation 2:1)

20 ul - digested TolC (~80 ng )
1.5 ul - digested vector [pSB] (~40 ng)
2.5 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________ 25 ul => TOTAL

=> SoxS (~103bp) and pSB1C3 (~2000bp) (ratio for ligation 3:1)

0.3 ul - H20
20 ul - digested SoxS (~88 ng )
1.2 ul - digested vector [pSB] (~29 ng)
2.5 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________