Assembly of invasion operon

Marcin

Task 1:

• Prepare chemocompetent bacteria

Detailed protocol

1. Incubate 100 ml of liquid bacterial culture until the OD value is 0.55 or little more
2. Incubate the bacteria on the ice for 15 minutes
3. Centrifuge the bacteria at 4°C for 15 minutes
4. Remove the supernatant and add 10 ml of 0.1 M solution of CaCl₂
5. Gently suspend the bacterial pellet and incubate the bacteria on the ice for 45 minutes
6. Centrifuge the bacteria at 4°C for 15 minutes
7. Remove the supernatant and add 2 to 5 ml of 0.1 M solution of CaCl₂
8. Prepate the aliquots of suspended bacteria which contain 100 μl of the solution
9. Store the bacteria in -80°C

Assembly of endosomal detection operon

Marcin

Task 1:

• Transformation of chemocompetent E. coli strain DH5α

Methods:

• Detailed protocol of ligation is described here. The only modification is total volume of ligation mixture prepared 20.07.09
• petri dish were hold in 37°C for 16 hours

Cloning of p53 coding sequence

Marcin

Task 1:

• Cloning p53 coding sequence to pKS plasmid

Methods:

• Ligation mixture composition: 10 μl digested p53, 25 μl digested pKS, 5 μl ligation buffer (Fermentas),8 μl MQ water, 2 μl ligase T4
• Duration of ligation was about 7 hours; reaction was conducted in 16 °c (approximately).

Task 2:

• Transformation of chemocompetent E. coli strain DH5α

Methods:

• Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes
• Detailed protocol of ligation is described here. The only modification is usage of half volume of ligation mixture prepared today/li>