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JULY: WEEK 3
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, 12thE9, 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.
Glycerol stocks were prepared for every culture, except for E11-1, E21-2, E23-2 because they were not sufficiently grown. July, 14thGlycerol 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:
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.
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.
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.
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Team:UNIPV-Pavia/Calendar/July/week3
From 2011.igem.org
