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...

Yet another attempt at obtaining a specyific Llo PCR product for fusion with the secretion signal

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, 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 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:

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