Cloning of p53 coding sequence

Marcin

Task 1:

• Prepare PCR reaction to amplify p53 coding sequence.

Methods:

• DNA template dilution:

1 μl 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 μl primer p53F (50 nM; Oligo.pl) 
0.5 μl primer p53R (50 nM; Oligo.pl) 
2.5 μl dNTPs (20 μM ;Fermentas) 
2 μl Pfu polymerase (EurX) 
5 μl Pfu Buffer (EurX) 
1 μl DNA template 
39 μl MQ water

• Negative control: the same as proper mixture 1, the only distinction is the lack of the DNA template.

• Program:

p53 (detailed destription is here)

Task 2:

• Prepare the bacterial cultures for isolation of plasmids containing ligated biobricks

Preparation of bacterial cultures

• Prepare LB medium with kanamycin
• Add one bacterial colony to 50 μl of medium
• Breed the bacteria about 12 hours