Team:Warsaw/Calendar-Main/18 July 2009

Plasmid digest mix was prepared as follows:

2μl Tango buffer (Fermentas)
1μl pKSII plasmid
0,5μl XbaI enzyme (Fermentas)
0,5μl SmaI enzyme (Fermentas)

The solution was topped up with H2O to the final volume of 20 μl.

Pho gene digest mix was prepared as follows:
```
2μl Tango buffer (Fermentas)
9μl purified gene
0,5μl XbaI enzyme (Fermentas)
```
The solution was topped up with H2O to the final volume of 20 μl.
The digest was kept for 3h at 37°C, and then the enzymes were inactivated for 20min. at 80°C.
The ligation mix was prepared as follows:
```
20μl plasmid
20μl gene
5μl ligation buffer with PEG 
1 μl T4 DNA ligase (Fermentas)
```
The solution was topped up with H2O to the final volume of 50 μl. The ligation was carried out in 18°C overnight (~15h)

Task:

Isolation of plasmid containing p53 CDS and digest them using PvuII restriction enzyme

Methods:

Plasmids weren isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here.
Digest of pKS/p53 plasmids using PvuII

Reaction mixture composition:

1 μl purified plasmid DNA product
0.5 μl PvuII (Fermentas)
2 μl Buffer Green (Fermentas)
16.5 μl MQ water

verification of the
restriction patterns

Electrophoresis condition:

voltage - 70V

time - 30 min

Comment:

The restriction patterns indicate that all isolated plasmids are empty

Franek

Tasks:

Transformation of Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a competent cells with BBa_K177024

Methods:

2 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing BBa_K177024 on pSB1A2 plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed with PlasmidMini set by A&A Biotechnology. The pellet from 5 ml of bacteria was used.

Chemocompetent E. coli cells from Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a strains were transformed according to our standard procedure with BBa_K177024

Results: