Team:Warsaw/Calendar-Main/18 July 2009

Insertion of the pho gene into the pKSII+ plasmid Kama  Plasmid digest mix was prepared as follows: 2&mu;l Tango buffer (Fermentas) 1&mu;l pKSII plasmid 0,5&mu;l XbaI enzyme (Fermentas) 0,5&mu;l SmaI enzyme (Fermentas) The solution was topped up with H2O to the final volume of 20 &mu;l. Pho gene digest mix was prepared as follows: 2&mu;l Tango buffer (Fermentas) 9&mu;l purified gene 0,5&mu;l XbaI enzyme (Fermentas) The solution was topped up with H2O to the final volume of 20 &mu;l. The digest was kept for 3h at 37&deg;C, and then the enzymes were inactivated for 20min. at 80&deg;C.

 The ligation mix was prepared as follows: 20&mu;l plasmid 20&mu;l gene 5&mu;l ligation buffer with PEG 1 &mu;l T4 DNA ligase (Fermentas) The solution was topped up with H2O to the final volume of 50 &mu;l. The ligation was carried out in 18&deg;C overnight (~15h)

Cloning of p53 coding sequence Marcin

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 <a href="http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf">here</a>.</li> Digest of pKS/p53 plasmids using PvuII</li> Reaction mixture composition: 1 &mu;l purified plasmid DNA product 0.5 &mu;l PvuII (Fermentas) 2 &mu;l Buffer Green (Fermentas) 16.5 &mu;l MQ water </li></ul> After the digest each plasmid solution was loaded into the 1% agarose gel</li></ul> <img src="http://2009.igem.org/wiki/images/5/58/PKS-kontrola_wstawki_18_07_09.png" height="35%" width="35%"> verification of the restriction patterns Electrophoresis condition: voltage - 70V time - 30 min  Comment:  The restriction patterns indicate that all isolated plasmids are empty

Testing different E. coli strains regarding lacI and AraC repressors
Franek

Tasks:


 * Alkaline lysis of bacterial cultures to obtain BBa_K177024 device


 * 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.

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


 * Will be determined by cell colonies presence on plates