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 for 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 4ng of purified BBa_B0032 DNA were transformed in MGZ1 in order to test the transformation efficiency. Plates were incubated ON at 37°C.
BMR Genomics sent results of parts sequencing; E17-2, E18-2, E31-1, E32-2, E33-2, E34-1, E35-2 were OK while E28-1 sequence was wrong.

July, 28th

Colonies grew in every plate. In fact E17-2, E18-2, E19-2 and E20-2, E37, E38, E39 and E40 showed grown colonies. We picked two colonies for each plate and inoculate them in 5 ml of LB + Cm 12.5. As regards to E41 and E42 parts, we resolved to let them still grow; in the late morning we picked and inoculate two colonies for both plates.
Plate with BBa_B0032 showed 9 colonies; the corresponding efficiency results in 2250 colonies/μg of DNA (very low, we need to prepare new competent MGZ1).

July, 29th

Glycerol stocks were prepared for E17-2, E18-2, E19-2, E20-2 in MGZ1.
Plasmid purification was carried out on over-night grown cultures (E41-1 did not grow!):

A 12x mix was prepared for screening digestions:

For each digestion 10 μl of purified DNA were used. Meanwhile a medium size gel was prepared.
In the afternoon gel electrophoresis was carried out:

Medium size gel

All clones were positive except for E42-2 and E41-2.
Glycerol stocks were prepared for E37-2, E38-1, E39-1, E40-2 and E42-1.
Ligations of consitutive promoter BBa_J23101 with different RBS in pSB4C5 were performed:

July, 30th

E28 and E41 were again transformed in 200 μl of MGZ1 competent cells.
J101-E5, J101-31 and J101-E7 ligations were transformed in 100 μl of TOP10 competent cells.

July, 31st

All plates showed colonies. One colony was picked from each of J101-E5, J101-31 and J101-E7 plates, inoculated in LB + Cm 12.5 and grown.