Team:Warsaw/Calendar-Main/2 August 2009

Cloning of p53 coding sequence Marcin Task 1:  Prepare PCR reaction to amplify p53 coding sequence.  Methods:  DNA template dilution:  1 &mu;l of plasmid solution (isolated in 11.07.09) was taken and diluted with 99 mi&;l of MQ water  PCR mixture composition: proper mixture 1: 0.5 &mu;l primer p53F (50 nM; Oligo.pl) 0.5 &mu;l primer p53R (50 nM; Oligo.pl) 2.5 &mu;l dNTPs (20 &mu;M ;Fermentas) 2 &mu;l Pfu polymerase (EurX) 5 &mu;l Pfu Buffer (EurX) 1 &mu;l DNA template 39 &mu;l MQ water Negative control: the same as proper mixture 1, the only distinction is the lack of the DNA template.   Program:</li></ul> p53 (detailed destription is <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/9_July_2009">here</a>) Task 2:  Prepare the bacterial cultures for isolation of plasmids containing ligated biobricks</li> </ul> Preparation of bacterial cultures  Prepare LB medium with kanamycin</li> Add one bacterial colony to 50 &mu;l of medium</li> Breed the bacteria about 12 hours</li></ul>