Team:Warsaw/Calendar-Main/16 September 2009

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Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil


Tasks:

  • pSB1A3 plasmid digest

Methods:

  • The digest mix was prepared as follows:

    5μl concentrated plasmid 
    1μl Buffer O (Fermentas)
    0,5μl EcoRI enzyme
    0,5μl 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:

  • pSB1A3 plasmid digest

Methods:

  • The digest mix was prepared as follows:

    5μl concentrated plasmid 
    1μl Tango Buffer (Fermentas)
    0,5μl XbaI enzyme
    0,5μl 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.

Contents

Assembly of fusion proteins

Marcin

Task 1:

  • Transformation of chemocompetent E. coli TOP10 strain

Construct to transform:

  • mitochondrial signal peptide CDS on pSB1A3 plasmid

Methods:

  • Detailed protocol of transformation is described here


Assembly of endosome detection module:

Marcin


Task 1: Prepare of BBa_K177044

Comment:

Due to some difficulties with preparing the BBa_K177037 I decided to create another biobrick, BBa_K177044 compose with following biobricks:

Methods:

  • Restriction digest of previously denoted biobricks:
    • Reaction mixture composition:
20 μl purified plasmid DNA product
1 μl XbaI (Fermentas), or 0.7 μ SpeI (Fermentas), the latter one was used to cut BBa_K177035
1 μl PstI (Fermentas)
5 μl Buffer Tango (Fermentas)
23 μl MQ water
  • Reactions were carried out 6 hours and were subsequently thermally inactivated.


Task 2: Gel-out of the previously described, digested biobricks

Methods:

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

Results:


Task 3: Ligation of the BBa_K177044 Methods:

  • Ligation 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)
  • Ligation were carried out 16 hours



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