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Team:UNICAMP-EMSE Brazil/Notebook/22 September 2011
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| - | + | '''Ligations:''' | |
| - | + | # IL-10 (~550bp) and pSB1C3 (~2000bp) (ratio for ligation 3:1) | |
| - | + | ||
| - | + | ||
| - | + | ||
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| - | + | ::*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 | |
| - | + | #Transformation (chemical shock protocol) | |
Latest revision as of 21:40, 23 September 2011
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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:
- 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
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
- Transformation (chemical shock protocol)
