Team:Warsaw/Calendar-Main/7 September 2009
From 2009.igem.org
Acquiring the internaline A gene from Listeria monocytogenes st. EDG-e genome
Jarek
Tasks:
- Electrophoretical segregation of PCR products from previous day in 0,8% agarosis gel (sample containing 10 microL was used).
- Purifing DNA from agarose block with A&A Gel-Out kit.
Results:
- Finally I have the correct PCR product (at least I hope it's that one).
Assembly of fusion proteins
Marcin
Task 1:
- Digest pKSII cloning vector using SmaI:
Methods:
- Reaction mixture composition:
15 μl purified plasmid DNA product 0.5 μl SmaI (Fermentas) 2 μl Buffer Tango (Fermentas) 12.5 μl MQ water
- Digestion was performed 6 hours and in next step enzyme was inactivated in 65 °C for 20 minutes
Task 2:
- Phosphorylation of previously purified sequence of mitochondrial signal peptide from Cox1 :
Methods:
- Reaction mixture composition:
15 μl purified plasmid DNA product 2 μl PNK Buffer (NEB) 0.5 μl PNK (NEB) 2.5 μl dNTPs mixture (EurX, concentration 5 mM)
- Reaction was performed 45 minutes and subsequently inactivate via heating (65 °C)
Task 3: Ligate of mitochondrial signal peptide to pKSII vector
- Methods:
- Ligation mixtures composition:
10 μl phosphorylated insert 8 μl digested vector 2.5 μl Tango buffer(Fermentas) 2.5 μl dNTPs mixture (EurX, concentration 5 mM) 1 μl ligase T4 (Fermentas)
- Ligation was carry out about 15 hours
Cloning of the mgtc promoter into the pSB1A3 plasmid
Kamil
Tasks:
- Ligation
- Bacteria transformation
Methods:
- The ligation mix was prepared as follows:
20μl purified plasmid backbone
10μl purified PCR product
4μl Ligase buffer (Fermentas)
1μl T4 Ligase (Fermentas)
5μl H2O- The ligation was carried out in 18°C for 3h and then inactivated in 80°C for 15 min.
- A fresh batch of chemocompetent bacteria was transformed with the ligation mix and incubated on agarose plates containing double dose of ampicilin.
Cloning of the cro-box into the pSB1A3 plasmidKamil
Tasks:
- Ligation to the plasmid
- Bacteria transformation
Methods:
- The ligation mix was prepared as follows:
10μl purified plasmid backbone
10μl purified PCR product
3μl Ligase buffer (Fermentas)
1μl T4 Ligase (Fermentas)
6μl H2O- The ligation was carried out in 18°C for 3h and then inactivated in 80°C for 15 min.
- A fresh batch of chemocompetent bacteria was transformed with the ligation mix and incubated on agarose plates containing double dose of ampicilin.
Inverting PLacI regulated parts of the switch and assembling the switch on a single plasmidAnia
Tasks:
April M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 May M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 June M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 July M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 August M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 September M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 October M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31