Team:Warsaw/Calendar-Main/11 July 2008

From 2008.igem.org

Revision as of 17:12, 26 September 2008 by Marcinoll (Talk | contribs)

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
next notebook entry

 



Preparation of constructs with OmpA protein fusions
1. Colony PCR on colonies from plates with transformations OmpA_alpha.
2. Confirmed transformant colonies inoculated to liquid LB with kanamycin.
Cloning of protein Z DNA to OmpA constructs
1. 2 colonies was inoculated to liquid LB broth with kanamycin

Preparation of construct omega-A

1. PCR A in 50 µl
template DNA - pKS-A4 1 µl
primer AP+NotI_N - 2 µl
primer AL+link10+homo2_N - 2 µl
Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl
dNTPs - 1 µl
Pfu turbo - 0.5 µl
H2o - 38.5 µl

program:
1. 95°C 3'
2. 95°C 30"
3.62°C 45"
4.72°C 45"
5.72°C 10'
6. keeping in 4°C

2. PCR omega in 50 µl
template DNA - pUC19 1 µl
primer OmegaLS - 2 µl
primer AOmegaPli - 2 µl
Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl
dNTPs - 1 µl
Pfu turbo - 0.5 µl
H2o - 38.5 µl

program: 1. 95°C 3'
2. 95°C 30"
3. 62°C 45"
4. 72°C 45"
5. 72°C 10'
6. keeping in 4°C
25 cycles
3. gel electrophoresis

4.reisolation from agarose gel

{{WarNotebookEnd}}