Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil

Tasks:

• PCR product digest

Methods:

• The digest mix was prepared as follows:

  40μl plasmid isolation
  5μl Buffer O (Fermentas)
  1μl EcoRI enzyme
  1μl PstI enzyme
  3μl H2O

• The digest was carried out in 37°C overnight and inactivated for 15 min. in 80°C.
• The product was later purified using the Montage PCR Centrifugal Filter Device.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil

Tasks:

• Creation of the cro-box double stranded DNA
• Cro-box digest

Methods:

• The mixture of equal volumes (10μl) of both ssDNA was heated to 94°C for 15min. and left to cool.
• The digest mix was prepared as follows:

  20μl plasmid isolation
  3μl Tango Buffer (Fermentas)
  1μl XbaI enzyme
  1μl PstI enzyme
  5μl H2O

• The digest was carried out in 37°C overnight and inactivated for 15 min. in 80°C.
• The product was later purified with dialysis.

Assembly of fusion proteins

Marcin

Task 1: Dialysis of PCR-amplified COX signal peptide

Comment:

Typical purification methods such as usage of gel-out are useless in the case of small fragments of DNA. It is the reason why I chose dialysis to purify the PCR product.

Methods:

• Dialysis were perform one hour
• After dialysis samples were elecrophoretically separated to investigate the identity and amount of DNA.

Results:

Task 2: Prepare the backbone plasmid pSB1K3 and COX mitochondrial signal to ligate them

• Reaction mixture composition:

20 μl purified plasmid DNA product (or PCR-amplified signal peptide)
1 μl PstI (Fermentas)
1 μl SpeI (Fermentas)
5 μl Buffer Tango (Fermentas)
23 μl MQ water

• Digestion was carry out 7 hours

Results:

Kanamycine-resistant plasmid must have been mistaken with some other construct because no biobrick is cut and the length of the plasmid is longer than expected. I decided to use pSB1A3.

Task 3: Ligation of mitochondrial signal peptide with pSB1A3

• Reaction mixture composition:

8 μl purified plasmid DNA product (or PCR-amplified signal peptide)
10 μl purified PCR product
2.5 μl dNTPs mixture (EurX, concentration 5 mM)
2.5 μl Buffer Tango (Fermentas)

• Ligation was carry out 15 hours