Team:UNICAMP-EMSE Brazil/Notebook/22 September 2011

Electrophoresis Gel 1, 22/09/2011.
Gel 1: IL-10 (A) / IL-10 (B) / TolC (A) / TolC (B)

22 September 2011

Sequences to ship

Quantification and checking digestion of sequences to ship

Objectives:

1. Digestion of IL-10 and TolC (pJ204 vector) to be gel purified and inserted in the pSB1C3 vector (standard backbone)

Samples:

• Gel 1:

• IL-10 (A) / IL-10 (B) / TolC (A) / TolC (B)

• Ladder:

• 1kB Plus DNA Ladder (Invitrogen)

• Gel Agarose concentration:

• 1,0%

• Result:

• All insert bands have the expected size

Electrophoresis Gel 2, 22/09/2011.
IL-10 (A) / IL-10 (B) / TolC (A) / TolC (B) - 1.5ul of purified DNA (each).

DNA purification from agarose gel and new electrophoresis to check

Samples:

• Gel 2:

• IL-10 (A) / IL-10 (B) / TolC (A) / TolC (B) - 1.5ul of purified DNA (each).

• Ladder:

• 1kB Plus DNA Ladder (Invitrogen)

• Gel Agarose concentration:

• 1,0%

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

0.8 ul - H20 9.4 ul - digested IL-10 (~150 ng ) 6.8 ul - digested vector [pSB] (~50 ng) 2 ul - 10X Ligation Buffer 1 ul - T4 Ligase _________________________________ 20 ul => TOTAL

______________________________________________________________ => TolC (~1600bp) and pSB1C3 (~2000bp) (ratio for ligation 2:1)

6.7 ul - H20 3.5 ul - digested TolC (~100 ng ) 6.8 ul - digested vector [pSB] (~50 ng) 2 ul - 10X Ligation Buffer 1 ul - T4 Ligase _________________________________ 30 ul => TOTAL