Team:Warsaw/Calendar-Main/16 September 2009
From 2009.igem.org
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 plasmidKamil
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_K177044Comment:
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 biobricksMethods:
- 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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