Cloning of the mgtc promoter into the pKSII+ plasmid

Kamil

Tasks:

• Plasmid assembly

Methods:

• The ligation mix was prepared as follows

  1ul plasmid, 3ul gene, 1ul ligation buffer B (Fermentas)
  1ul T4 DNA ligase (Fermentas)
  1ul 30% PEG, 1ul 10mM ATP

  The solution was topped up with H2O to the final volume of 10 ul. The ligation was carried out in 37°C for 1h and then inactivated for 15min. at 65°C.

• A 200ul batch of chemocompetent bacteria was transformed with 3ul of ligation mix and incubated on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG.

UPDATE: No luck this time, back to the drawing board...

Making Llo fusion with secretion signal peptide

Kuba

PCR was preformed on a product of classical biobrick-format Llo (reamplification)

three reactions were preformed, each with increasing concentration of template

• Methods:

  • PCR mixture's composition:

    2,5ul pfu buffer (Fermentas)
    2,5ul MgSO4 (Fermentas)
    1,5ul primers
    1,5ul dNTPs (10 mM)
    0,5ul pfu turbo polymerase
    1ul template DNA from Listeria

    the solution was topped up with H₂O to 25ul.

    One of the samples also contained 1 ul of DMSO

  • PCR programs:

   4min 95°C 
  (30s 95°C, 40s 41°C, 1min40s 72°C)x3 
  (30s 95°C, 40s 44°C, 1min40s 72°C)x28  10min 72°C 
  ~ 7°C

  • Electrophoretic separation on 1% agarose gel
  
  

  Results:

  
  • Gel (from left)

1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
2. sample no.1 (1ul of 100-fold diluted template)
3. sample no. 2 (2ul of 100-fold diluted template)
4. sample no. 3 (1ul of template)

no significant amounts of desired product were obtained

Cloning of p53 coding sequence

Marcin

Tasks:

• ligation of p53 coding sequence with pKS plasmid digested by XbaI and SmaI.

Methods:

• Ligation mixture composition:

  13 ul digested p53
  5 ul Ligase Buffer (Invitrogen)
  1 ul digested pKS
  1 ul ligase T4 (Fermentas)

• logation program:

digest

 7h 14°C 
 15 min 80°C 
 ~4°C
 

Tasks:

• Transformation of chemocompetent E. coli strain DH5alpha.

Methods:

• thaw bacteria on the ice - 10 minuts
• add 10 ul of ligation mixture to the bacteria
• incubation on the ice - 20 minutes
• heat shock - 1 minut, 42°C
• incubation on the ice - 2 minuts
• add 800 ul of SOB medium to the bacteria
• incubation in 37°C - 1 h
• Plating on selective LB medium supplemented with ampicillin, X-Gal and IPTG.

Ligation was unsuccesful, there was no bacterial colonies on the plates.