Assembly of endosomal detection operon

Marcin

Task 1:

• Digest of isolate plasmids with ligated biobricks to verify the success of ligation

Methods:

• Digest of isolate plasmids using XbaI and PstI
• Reaction mixture composition:

  0.5 μl purified plasmid DNA product 
  0.5 μl XbaI (Fermentas)
  0.5 μl PstI (Fermentas) 
  2 μl Buffer Tango (Fermentas) 
  16.5 μl MQ water

• The reaction was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.
• In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids

Comment:

All isolated plasmids have not insert. The cloning must be performed another time

Task 2:

• Restriction digest of biobricks

Comment:

Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested:

BBa_B0032

BBa_c0040

BBa_C0051

BBa_E0032

Methods:

• Digest of BBa_B0032 using SpeI and PstI
• Reaction mixture composition:

  10 μl purified plasmid DNA product 
  0.5 μl SpeI (Fermentas)
  1 μl PstI (Fermentas) 
  5 μl Buffer Tango (Fermentas) 
  34 μl MQ water

• Digest of other biobricks using PstI and XbaI
• Reaction mixture composition:

  10 μl purified plasmid DNA product 
  1 μl XbaI (Fermentas)
  1 μl PstI (Fermentas) 
  5 μl Buffer Tango (Fermentas) 
  34 μl MQ water

• Both reaction were performed approximately 7 hours they were subsequently inactivated via heating in 80°C for 20 minutes

Task 3:

• Ligations of biobricks

Methods:

• Ligation mixture composition:

  8 μl digested plasmid with RBS 
  9 μl digested CDS 
  2 μl ligation buffer (Fermentas, PEG4000 have been added previously) 
  1 μl ligase T4 (Fermentas)

• Negative control mixture composition: 8 μl digested plasmid with RBS 2 μl ligation buffer (Fermentas, PEG4000 have been added previously) 1 μl ligase T4 (Fermentas) 9 μl MQ water
• Duration of ligation was about 12 hours

Construction of K177012 operon1_part2

Ania

Tasks:

• Gel electrophoresis of the obtained clones.
• Repeat the ligation of the PcI and RBs.3LacI parts.

Results:

• Only small amount of DNA visible on the gel in 4 samples out of 20. Probably plasmid isolation has been done using too old solution 2. The visible DNA fragments are not the correct clone.