Acquiring the internaline A gene from Listeria monocytogenes st. EDG-e genome

Jarek

Tasks:

• Digestion of PCR products, separetly with MboI and CaiI restriction endonucleases
• Electrophoretical segregation of digested samples in 1,5% agarose gel
• Preparation of another PCR reaction with L.monocytogenes genome DNA

Results:

• There were no visible products of digestion, even after long treatment with ethidium bromide. Propably the Clean-Up kit isn't working well. I'll try to acquire IntA gene with another PCR.

Preparation of glycerol stocks

• Preparation of glycerol stocks following the prodcedure described here

pAraC + GFP

plac + RBS + cI + ter

plac + RBS + cI + RBS + GFP + ter

J07037

plac

pSB6A1

Assembly of endosomal detection operon

Marcin

Task 1:

• Isolate the plasmids containing following construct:
• BBa_C0051 with BBa_B0032 on pSB1A3 plasmid

Methods: Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described[http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf here]

Task 2:

• Digestion of plasmids previously described constructs

Methods:

• Reaction mixture composition:

20 μl purified plasmid DNA product
1 μl XbaI (Fermentas)
1 μl PstI (Fermentas) 
2 μl Buffer Tango (Fermentas)
15 μl MQ water

• Digest was performed about six hours and subsequently thermal inactivated

Task 3:

• Gel-outs of construct described in Task 2

Methods:

• All gel-outs were performed using the EurX gel-out kit according to the manual

Task 4:

• Ligation of following constructs:
  • [http://partsregistry.org/Part:BBa_B0032BBa_B0032] with [http://partsregistry.org/Part:BBa_E0022BBa_E0022] connected to [http://partsregistry.org/Part:BBa_B0032BBa_B0032] with [http://partsregistry.org/Part:BBa_C0051BBa_C0051] on [http://partsregistry.org/Part:pSB1A3pSB1A3] plasmid
  • cro CDS on [http://partsregistry.org/Part:pSB1A3pSB1A3]
  • Bax CDS on [http://partsregistry.org/Part:pSB1A3pSB1A3]

Methods:

• Reaction mixture composition:

10 μl digested insert
8 μl digested vector 
2 μl Tango buffer(Fermentas)
3 μl dNTPs mixture (EurX, concentration 5 mM) 
1 μl ligase T4 (Fermentas)

• Reaction were carried out 12 hours and subsequently thermally inactivated.

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:

• [Nice gel picture of empty vectors here]

Conclusions:

• Respawn and try again in... 3 days.

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 1% agarose gel

Results:

• [Nice gel picture of empty vectors here]

Conclusions:

• Respawn and try again in... 3 days.