Assembly of fusion proteins

Marcin

Task 1:

• Digest plasmids isolated in 09.09.2009 to reveal the identity of the constructs

Methods:

• Reaction mixture composition:

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

• Digest was performed 90 minutes

Results:

• None constructs are correct. It is obligated to perform cloning second time. If I dephosphorylate the vector prior to ligation the result should be better.

Task 2:

• dephosphorylation the following molecules previously digested by resctriction enzymes:
  • [http://partsregistry.org/Part:BBa_K177036BBa_K177036]
  • pKSII cloning vector
• Reaction mixture composition:

15 μl purified plasmid DNA product
2 μl Tango Buffer (Fermentas)
1 μl CIAP (Fermentas)

• Reaction was performed 45 minutes and subsequently inactivate via heating (65 °C)

Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil

Tasks:

• Plasmid isolation
• Control digest

Methods:

• Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).
• The digest mix was prepared as follows:

  20μl isolated plasmid
  3μl Buffer O (Fermentas)
  1μl PstI enzyme
  1μl EcoRI enzyme
  5μl H2O

• The digest was carried out for 3h in 37°C and then inactivated in 80°C for 15min.
• The results were visualised on 1% agarose gel

Results:

• All empty

Conclusions:

• So tired, need sleep...

Cloning of the cro-box into the pSB1A3 plasmid

Kamil

Tasks:

• Plasmid isolation
• Control digest

Methods:

• Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).
• The digest mix was prepared as follows:

  20μl isolated plasmid
  3μl Buffer O (Fermentas)
  1μl PstI enzyme
  1μl EcoRI enzyme
  5μl H2O

• The digest was carried out for 3h in 37°C and then inactivated in 80°C for 15min.
• The results were visualised on 2% agarose gel

Results:

• All empty

Conclusions:

• So tired, need sleep...