Team:UNIPV-Pavia/Calendar/July/settimana5

July, 25th

Red colonies were observed from E36 plate, suggesting that the transformation was successful. A colony was picked form the plate.

Digestions of previously purified plasmids were performed for ligations:

Reactions were incubated at 37°C for three hours while a small size agarose gel was prepared according to protocols.
In the afternoon gel electrophoresis was performed:

Small size gel

After gel extraction, digested DNA was quantified:

Ligations were performed:

Inoculum of E17-2, E18-2, E19-2 or E20-2 to amplify DNA and transfer it into MGZ1; refill for E36 liquid culture.
Glycerol stock fpr E36 was prepared
250 ml of LB + Cm 12.5 were prepared.

July, 26th

Plasmids containing E17-2, E18-2, E19-2, E20-2 and E36 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:

Digestion of E36 was performed for ligations:

The sample was incubated at 37°C for three hours while a small-size agarose gel was prepared; in the afteroon gel electrophoresis was carried out:

Small size gel

After gel extraction, digested DNA was quantified:

Then ligations were performed in a final volume of 10 μl:

Ligations were incubated at 16°C ON.

July, 27th

E37, E38, E39, E40, E41, E42, E17, E18, E19 and E20 were transformed in 100 μl of MGZ1 competent cells according to protocols. Moreover a plasmid containing BBa_B0032 was transformed in order to test the transformation efficiency. Plates were incubated ON at 37°C.

July, 28th

Colonies grew in every plate. Infact both 17-2, E18-2, E19-2 and E20-2 parts, whose DNA was recovered after MiniPrep extraction kit, both E37, E38, E39 and E40 parts showed grown colonies. We picked two colonies from all parts. As regards to E41 and E42 parts, we resolved to let them still grow.
Plate with BBa_B0032 showed 9 colonies. The corresponding efficiency results 9/4 [1/ng] = 2250 [1/μl]