Team:Warsaw/Calendar-Main/31 August 2009
From 2009.igem.org
Cloning of the mgtc promoter into the pSB1A3 plasmid
Kamil
Tasks:
- mgtC promoter digest
- Ligation
Methods:
- The digest mix was prepared as follows:
40μl purified PCR product
5μl Buffer O (Fermentas)
1μl EcoRI enzyme
1μl PstI enzyme
3μl H2O- The digest was carried out in 37°C for 3h and inactivated for 15 min. in 80°C.
- The product was later purified using Montage PCR Centrifugal Filter Device (Milipore).
- The ligation mix was prepared as follows:
40μl purified plasmid backbone
20μl purified PCR product
8μl Tango Buffer (Fermentas)
5μl 5mM dNTPs (EURx)
1μl T4 Ligase (Fermentas)
6μl H2O- The ligation was carried out in 18°C overnight and then inactivated in 80°C for 15 min.
Cloning of the cro-box into the pSB1A3 plasmidKamil
Tasks:
- Ligation to the plasmid
Methods:
- The ligation mix was prepared as follows:
40μl purified plasmid backbone
20μl Cro-Box
8μl Tango Buffer (Fermentas)
5μl 5mM dNTPs (EURx)
1μl T4 Ligase (Fermentas)
6μl H2O- The ligation was carried out in 18°C overnight and then inactivated in 80°C for 15 min.
Making of the plac-RBS-llo-intA partJarek
Tasks:
- Preparing the liquid cultures from colonies that grew after transformation and incubationg them in 37C degree.
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