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 endosomal detection operon

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