Team:UNIPV-Pavia/Calendar/July/settimana3

July, 11th

Digestions of previously purified plasmids were performed for ligations:

Reactions were incubated at 37°C for three hours while two medium-size agarose gels were prepared according to protocols.

In the afternoon gel electrophoresis was performed.

Medium size gel

Medium size gel

As shown, in figure all clones were positive, so we cut and purified the bands of interest.

After gel extraction, digested DNA was quantified:

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

Ligations were incubated ON at 16°C.

July, 12th

E9, E10, E11, E12, E14, E15, E17, E18, E19, E20, E21, E22 and E23 were transformed in 100 μl of TOP10 competent cells according to protocols, while E13 and E16 ligations were transformed in 100 μl of MGZ1 competent cells. Plates were incubated ON at 37°C.

July, 13th

All plates showed a lot of colonies, except for E13, E16 (one colony),while for E14, E15 no colonies were observed (probably the parts gave too high metabolic burden that prevents cells growth or because of low transformation efficiency of MGZ1 strain). Because of RFP, E21 was red, E22 and E23 a little bit less. Two colonies (where possible) were picked for each plate.
500 ml of LB with Ampicillin were prepared.
Another ligation was performed:

July, 14th

Glycerol stocks for E11-1, E21-2 and E23-2 were prepared. Cultures grew overnight; plasmid purification and quantification were carried out:

A 25 x mix was prepared in order to perform PCR on E9-1, E9-2, E10-1, E10-2, E11-1, E11-2, E12-1, E12-2, E17-1, E17-2, E18-1, E18-2, E19-1, E19-2, E20-1, E20-2, E21-1, E21-2, E22-1, E22-2, E23-1, E23-2:

24 μl were aliquoted in each tube, together with 1 μl of each liquid culture. PCR cycles parameters were set as follows:

• 94°C 30 seconds (denaturing)
• 60°C 1 minute (annealing)
• 72°C 2 minutes (elongation)

In order to screen the DNA of the only two colonies of E13 and E16, a digestion for each ligation was performed:

Reactions were incubated at 37°C for three hours.
BBa_B0032 was transformed in 100 μl of MGZ1 competent cells to test if there was any problem with their transformation efficiency, while BBa_R0040 in BBa_J61002 was transformed in 200 μl of TOP10 competent cells. Plates were incubated ON at 37°C.
Two medium size gels were prepared.

In the afternoon gel electrophoresis was performed:

Medium size gel (20 wells)

Medium size gel

As shown in figure, all clones were positive, except for E9-1, E17-1, E17-2, E18-1, E18-2, E19-1 which didn't show any DNA band. E9-2, E17-2, E18-2, E19-2, E20-2, E21-1, E22-2, E23-1 plasmid purification was carried out, in order to prepare samples for sequencing.

E9-2, E10-1, E11-1, E12-1, E13-1, E16-1, E19-2, E20-2, E21-1, E22-2 and E23-1 glycerol stocks were preserved, while the others were thrown away. Cells harbouring E9-2, E10-1, E11-1, E12-1, E13-1, E16-1, E21-1, E22-2, E23-1 were inoculated in 5 ml LB + Amp for next week ligations.

July, 15th

The plate containing MGZ1 transformed with BBa_B0032 showed a lot of colonies with satellite cells; a colony was picked for BBa_R0040 in BBa_J61002 as the plate showed red colonies and it was inoculated in 5 ml of LB+Amp and gorwn at room temperature.
Liquid cultures grew until saturation.

In order to screen again E17-1, E17-2, E18-1 and E18-2 ligations, digestions with EcoRI and PstI endonucleases were carried out:

Digestions were incubated at 37°C for three hours, while a small size gel was prepared; in the afternoon gel electrophoresis was carried out:

Small size gel

As three out of four clones were correct, E17-2 and E18-2 were kept.
E9-2, E10-1, E11-1, E12-1, E13-1, E16-1, E19-2, E20-2, E21-1, E 22-2 and E23-1 DNA was sent to BMR genomics for sequencing.
Plasmid purification was performed on liquid cultures: