Assembly of endosomal detection operon

Marcin

Task 1:

• Ligations of biobricks

Comment:

Due to obtain set of biobricks which each of them contain RBS and particular coding sequence I designed the following elemets (the order of biobricks is from the 5'end to 3' end):

1. BBa_B0032+BBa_c0040
2. BBa_B0032+BBa_C0051
3. BBa_B0032+BBa_E0032
4. BBa_B0032 (ligation of empty vector as a negative control of the experiment)

Methods:

• Ligation mixture composition: 3 μl digested plasmid with RBS, 3 μl digested CDS, 5 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4, 13 μl MQ water
• Negative control mixture composition: 3 μl digested plasmid with RBS, 5 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4, 16 μl MQ water
• Duration of ligation was about 12 hours

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 total volume of ligation mixture prepared 16.07.09
• petri dish were hold in 37°C for 36 hours

Cloning of p53 coding sequence

Marcin

Task:

• Isolation and evaluation of pKS/p53 plasmid

Besides high effectivity of transformation all of the bacterial colonies were blue. It is possible that excess of X-Gal may cause some problems with the selection system. Due to this uncentainty I decide to prepare a few bacterial breedings to isolate the plasmids and digest them.

Methods:

1. Prepare LB medium with kanamycin
2. Add 3.5 ml of the medium to the probes
3. Add one bacterial colony to each probe
4. Breed the bacteria about 8 hours